STANDARD MEASUREMENTS, DATA, APPENDIX 1 AND ABBREVIATIONS Common Abbreviations APPENDIX 1A

A260 absorbance at 260 nm AUFS absorbance units, full scale A adenine or adenosine; one-letter code for AUS Arthrobacter ureafaciens alanine β-gal β-galactosidase Ab antibody BAC bacterial artificial chromosome; ABTS [2,2-azino-di(3-ethylbenzothiazoline biospecific affinity chromatography sulfonate)] BAP bacterial alkaline phosphatase acetyl CoA acetyl coenzyme A BBS BES-buffered solution; borate-buffered AcMNPV Autographica californica saline multiply-enveloped nuclear polyhedrosis BCIP 5-bromo-4-chloro-3-indolyl phosphate virus BDB bis-diazobenzidine ADA adenosine deaminase BES N,N-bis(2-hydroxyethyl)-2- ADC analog-to-digital converter aminoethanesulfonic acid ADH alcohol dehydrogenase BHI brain heart infusion (medium) ADP adenosine 5-diphosphate biotin-11-dUTP 8-(2,4-dinitrophenyl-2,6- ADSL asynchronous digital subscriber line aminohexyl)aminoadenosine-5- AEC 3-amino-9-ethylcarbazole triphosphate or 2-deoxyuridine-5- AES 3-aminopropyltriethoxysilane triphosphate-5-allylamin biotin AEX anion exchange bis; bisacrylamide N,N-methylene AFLP amplified fragment length bisacrylamide polymorphism bis-Tris 2-bis(2-hydroxyethyl)amino-2- Ag antigen (hydroxymethyl)-1,3-propanediol AIDS acquired immune deficiency syndrome BLAST Basic Local Alignment Research AK adenosine kinase Tool ALARA as low as reasonably achievable Bluo-gal indoyl-β-D-galactopyranoside ALPS autoimmune/lymphoproliferative BMP bitmap (file format) syndrome Boc t-butyloxycarbonyl AM acetomethyl (moiety) BOP benzotriazolyl-N-oxy- AMan anhydro-D-mannose tris(dimethylamino)phosphonium AMP adenosine 5-monophosphate hexafluorophosphate AMPPD disodium bp base pair 3,4-methoxyspiro({1,2-dioxetane-3,2- BPV bovine papilloma virus tricyclo[3.3.1.13,7]decan})phenyl Bq Becquerel phosphate BrdU 5-bromodeoxyuridine AMV avian myeloblastosis virus BS3 bis(sulfosuccinimidyl) suberate ANOVA analysis of variance BSA bovine serum albumin AP alkaline phosphatase; apyrimidinic BSL biosafety level (sites) Bst Bacillus stearothermophilus DNA APH aminoglycoside phosphotransferase (polymerase) APHIS Animal and Plant Health Inspection C cytosine or cytidine; one-letter code for Service cysteine aPKC atypical protein kinase C C16TAB hexadecyl trimethylammonium Apr ampicillin resistant bromide APRT adenosine phosphoribosyltransferase CA3 chromomycin A3 APS ammonium persulfate CAD carbamoylphosphate synthetase ARS autonomous replication sequences CaM calmodulin ASPECT augmented surface polyethylene cAMP adenosine 3,5-cyclic-monophosphate prepared by chemical transformation cA-PrK cyclic AMP-dependent protein ATA aurintricarboxylic acid kinase Standard ATCC American Type Culture Collection CAPS [cyclohexylamino]-1-propanesulfonic Measurements, ATP adenosine 5 -triphosphate acid Data, and Abbreviations Current Protocols in Molecular Biology (2005) A.1A.1-A.1A.8 A.1A.1 Copyright C 2005 by John Wiley and Sons, Inc. Supplement 70 CAT chloramphenicol acetyltransferase CTC charge-transfer chromatography CATH Class, Architecture, Topology, and CTD C-terminal domain Homologous superfamily CTP cytidine 5-triphosphate CCD charge-coupled device CWS cell wall skeleton CCR Coriell Cell Repository CXA contextual expression analysis CD cluster of differentiation (antigens); D dextrorotatory circular dichroism dA deoxyadenosine CDC Centers for Disease Control Da Dalton cDNA complementary deoxyribonucleic DAB 3,3-diaminobenzidine acid DABCO 1,4-diazobicyclo-[2.2.2]octane CD-ORD circular dichroism–optical DAD diode array detection rotatory dispersion DAG diacylglycerol CDP cytidine 5-diphosphate dAMP deoxyadenosine monophosphate CD-ROM compact disk read-only memory DAPI 4,6-diamidino-2-phenylindole CDS coding sequence d(A-T) deoxyadenylate-deoxythymidylate CE capillary electrophoresis dATP deoxyadenosine triphosphate CED 3 cyanoethyl protected DBE direct blotting electrophoresis CEF chicken embryo fibroblast DBM diazobenzyloxymethyl CERN European Nuclear Research Council dC deoxycytosine CEX cation exchange DCA dichloroacetic acid CFA complete Freund’s adjuvant DCC dextran-coated charcoal; CFU colony-forming unit N,N-dicyclohexylcarbodiimide CGH comparative genome hybridization dCF 2-deoxycoformycin CHAPS 3-[(3-cholamidopropyl)- dCMP deoxycytidine monophosphate dimethylammonio]-1-propane-sulfonate dCTP deoxycytidine triphosphate CHEF contour-clamped homogeneous DD differential display electric field ddATP dideoxyadenosine triphosphate CHES 2-(N- DDBJ DNA Data Bank of Japan cyclohexylamino)ethanesulfonic ddCTP dideoxycytidine triphosphate acid DDDS distributed document delivery system CHO Chinese hamster ovary (cells) ddGTP dideoxyguanosine triphosphate Ci curie ddNTP dideoxynucleoside triphosphate CID charge-injection device; ddTTP dideoxythymidine triphosphate collision-induced dissociation DEA diethylamine CIP calf intestine phosphatase DEAE diethylaminoethyl CLEAR cross-linked acrylate ethoxylate DEPC diethylpyrocarbonate resin DES diethylstilbestrol cM centimorgans df degrees of freedom CM complete minimal (medium); dG deoxyguanosine carboxymethyl dGTP deoxyguanosine triphosphate CMC critical micelle concentration DHFR dihydrofolate reductase CML chronic myelogenous leukemia DIEA N,N-diisopropylethylamine CMP cytidine 5-monophosphate DIGE difference gel electrophoresis Cmr chloramphenicol resistant DiI 1,1-dihexyl-3,3,3,3- CMV cytomegalovirus tetramethylindocarbocyanine perchlorate 4CN 4-chloro-1-napthol DIP Database of Interacting Proteins; CNBr cyanogen bromide deletion-insertion polymorphism Con A concanavalin A dITP deoxyinosine 5-triphosphate CORT cloning of receptor targets DMB 1,2-diamino-4,5- CPC cetylpyridinium chloride methylenedioxybenzene CPG control pore glass dihydrochloride cpm counts per minute DMEM Dulbecco’s modified Eagle (or CRD cross-reacting determinant minimum essential) medium CS chemical sequencing DMF dimethylformamide CSPD disodium 3-(4-methoxyspiro[1,2- DMS dimethyl sulfate dioxetane-3,2-(5-chloro)tricyclo DMSO dimethyl sulfoxide Common [3.3.1.13,7]decan]-4-yl)phenyl phosphate DMT dimethoxytrityl Abbreviations CTAB cetyltrimethylammonium bromide DNA deoxyribonucleic acid A.1A.2

Supplement 70 Current Protocols in Molecular Biology DNase deoxyribonuclease FISH fluorescence in situ hybridization DNP 2,4-dinitrophenyl Fmoc fluorenylmethyloxycarbonyl dNTP deoxynucleoside triphosphate FOA fluoroorotic acid DOTMA n-[1-(2,3-dioleoyloxy)propyl]- FPLC fast protein; fast peptide; or fast N,N,N,-trimethylammonium chloride polynucleotide liquid chromatography DPA diphenylamine FQDN fully-qualified domain name dpm disintegrations per minute FRET fluorescent resonant energy transfer ds double-stranded FSC forward (light) scatter (in flow DSA Datura stramonium agglutinin cytometry) DSG disuccinimidyl glutarate FSSP Fold classification based on DSL digital subscriber line Structure-Structure alignment of Proteins DSS disuccinimidyl suberate FTIR Fourier transform infrared dT deoxythymidine (spectroscopy) DTE direct transfer electrophoresis FTP File Transfer Protocol DTT dithiothreitol Fuc L-fucose dTTP deoxythymidine triphosphate FUdR 5-fluoro-2-deoxyuridine dUMP deoxyuridine monophosphate g gravity (unit of centrifugal force) dUTP deoxyuridine triphosphate G gauge; guanine or guanosine; one-letter DvB deleted V (Mls-like loci) code for glycine EBI European Biotechnology Information GAG glycosaminoglycan EBV Epstein-Barr virus Gal D-galactose EC embryonic carcinoma; Enzyme GalNAc N-acetylgalactosamine Commission GALV gibbon ape leukemia virus ECL enhanced chemiluminescence GANC gancyclovir ECTEOLA epichlorohydrin triethanolamine Gb gigabyte EDC N-ethyl-N-[(dimethylamino) propyl] GDB Genetic Data Base carbodiimide hydrochloride GDP guanosine 5-diphosphate EDTA ethylenediaminetetraacetic acid GEM gene expression monitoring EGCase endoglycoceramidase GF gel filtration (chromatography) EGFR epidermal growth factor receptor GFP green fluorescent protein EGTA ethylene glycol-bis(β-aminoethyl GIF graphics interchange (file) format ether)-N,N,N,N-tetraacetic acid Glc D-glucose ELISA enzyme-linked immunosorbent assay GlcA D-glucuronic acid EMBL European Molecular Biology GLC-FID gas-liquid chromatography with Laboratory flame ionization detection EMC encephalomyocarditis GLC-MS gas-liquid chromatography with EMCV encephalomyocarditis virus mass spectroscopic detection EMS ethyl methanesulfonate GlcN D-glucosamine EMSA electrophoretic mobility shift assay GlcNAc N-acetylglucosamine Endo endoglycosidase GlUA; GlA D-glucuronic acid EOF electroosmotic flow GM-CSF granulocyte/macrophage colony EP-PCR error-prone polymerase chain stimulating factor reaction GMP guanosine monophosphate ES embryonic stem (cells) GMS genomic mismatch scanning ESI electrospray ionization (mass GNA Galanthus nivalus agglutinin spectrometry) GPI glycosyl phosphatidyl inositol EST expressed sequence tag GRAIL Gene Recognition and Analysis ET energy transfer Internet Link exo exonuclease GRASS Graphical Representation and F Farad Analysis of Structure Server FACS fluorescence-activated cell sorting GS glutamine synthetase FAQ frequently asked questions GSS genome survey sequence FBS fetal bovine serum GST glutathione S-transferase FCS fetal calf serum gTLD generic top-level domains FITC fluorescein isothiocyanate GTP guanosine 5 -triphosphate Standard FFPE formalin-fixed paraffin-embedded GUI graphical user interface Measurements, (tissue) GUS β-glucuronidase Data, and Abbreviations FIGE field-inversion gel electrophoresis Gy Gray (radioactivity unit) A.1A.3

Current Protocols in Molecular Biology Supplement 70 HA (influenza) hemagglutinin protein HRPO horseradish peroxidase HAT hypoxanthine/aminopterin/thymidine hsiRNA heterochromatic short interfering (medium) RNA HATU O-(7-azabenzotriazol-1-yl)- HS-TBST high-salt TBST (buffer) N,N,N,N-tetramethyluronium HSV herpes simplex virus hexafluorophosphate HTG/HTGS high-throughput genome HBSS Hanks’ buffered salt solution sequence HBTU O-benzotriazol-1-yl-N,N,N,N- HTML hypertext markup language tetramethyluronium hexafluorophosphate Hz hertz HCG human chorionic gonadotropin IAA 3-β indoleacrylic acid; indole-3-acetic hCMV human cytomegalovirus acid HeBS HEPES-buffered saline IACUC Institutional Animal Care and Use HEC hydroxyethylcellulose Committee HEPA high-efficiency particulate air (filter) ICAT isotope coded affinity tagging HEPES N-2-hydroxyethylpiperazine-N-2- i.d. inner diameter ethanesulfonic acid IdoA; IdUA; IdA L-iduronic acid HFBA heptafluorobutyric acid IEF isoelectric focusing hGH human growth hormone IEX ion exchange HGPRT hypoxanthine-guanine Ig immunoglobulin phosphoribosyltransferase imm immunity region HIC hydrophobic interaction IMAC immobilized metal affinity chromatography chromatography HILIC hydrophilic interaction IMPDH inosine-monophosphate chromatography dehydrogenase HIV human immunodeficiency virus IODOGEN 1,3,4,6-tetrachloro-3α,6α- HOAt 1-hydroxy-7-azabenzotriazole diphenylglycouril HOBt 1-hydroxybenzotriazole IP Internet Protocol HPAE-PAD high-performance anion IPTG isopropyl-1-thio-β-D-galactoside exchange chromatography with pulsed IR infrared amperometric detection IRES internal ribosomal entry site HP-BAC high-performance biospecific/ ISDN integrated services digital network biomimetic affinity chromatography ISH in situ hybridization HPCF high-performance chromatofocusing ISP Internet service provider HP-CTC high-performance charge transfer ISPCR in situ PCR chromatography IVT in vitro transcription HPH hygromycin-B-phosphotransferase JIPID Japan International Protein HP-HIC high-performance Information Database hydrophobic-interaction chromatography JPEG Joint Photographic Experts Group HP-HILIC high-performance hydrophilic (file format) interaction chromatography K Michaelis constant HP-IEX high-performance ion-exchange kb kilobase chromatography kbps kilobits per second HP-IMAC high-performance immobilized Kd dissociation constant metal ion affinity chromatography kDa kilodalton HPLC high-performance liquid KEGG Kyoto Encyclopedia of Genes and chromatography Genomes HP-LEC high-performance ligand-exchange KHz kilohertz chromatography KLH keyhole limpet hemocyanin HPMC hydroxypropyl methyl cellulose Kmr kanamycin resistant HP-MMC high-performance mixed mode L levorotatory chromatography LAMP lysosome-associated protein HP-NPC high-performance normal phase LAN local area network chromatography LB Luria-Bertani (medium) HPRT hypoxathine-guanine LC liquid chromatography phosphoribosyltransferase LCL lymphoblastoid cell lines HP-SEC high-performance size-exclusion LCM laser capture microdissection Common chromatography LCV lymphocryptovirus Abbreviations A.1A.4

Supplement 70 Current Protocols in Molecular Biology LEC ligand-exchange chromatography MMDM Molecular Modeling Database of LIF leukemia inhibitory factor; NCBI laser-induced fluorescence (detector) MMLV Moloney murine leukemia virus LMPCR ligation-mediated polymerase MMT monomethoxytrityl chain reaction MMTV mouse mammary tumor virus LPA linear polyacrylamide mmu millimass unit or one thousandth of a LRSC lissamine rhodamine Dalton LTR long terminal repeat MNase micrococcal nuclease Lumigen-PPD 4-methoxy-4-(3-phosphate MOI multiplicity of infection phenyl)-spiro-[1,2-dioxetane- MolMovDB Database of Macromolecular 3,2(-adamantane)], disodium salt Movement LysoPC lysophosphatidylcholine MoMuLV Moloney murine leukemia virus µF microfarad MOPS 3-(N-morpholino)propane sulfonic M relative molecular weight acid mA milliampere mp melting point MAA Maackia amurensis agglutinin MPA mycophenolic acid MAb, mAb monoclonal antibody MPC magnetic plate chamber MAB maleic acid (buffer) MPSS Massive Parallel Signature MALDI matrix-assisted laser Sequencing desorption/ionization (mass spectrometry) mRNA messenger ribonucleic acid MALDI-TOF matrix-assisted laser MS mass spectroscopy desorption/ionization time-of-flight (mass MSCV murine stem cell virus spectroscopy) MS/MS tandem mass spectrometry Man D-mannose MSX methionine sulfoximine MAP mitogen-activated protein; multiple Mtv mammary tumor virus designation antigenic peptide MTX methotrexate Mb megabase, megabyte MUG 4-methylumbelliferyl-β-D-galactoside Mbp megabase pair MUP methylumbelliferyl phosphate MBP maltose-binding protein MVA Modified vaccinia virus Ankara Mbps megabits per second MWCO molecular weight cutoff MBS m-maleimidobenzyl-N- NA not applicable hyderoxysuccinimide ester NAD nicotinamide adenine dinucleotide MBTH 3-methyl-2-benzothiazolinone Na-DOC sodium deoxycholate hydrazone hydrochloride NBF neutral buffered formalin MCA methyl celluloacetate ether NBRF National Biomedical Research MCAC metal-chelate affinity Foundation chromatography NBT nitroblue tetrazolium MCS multiple cloning site NCAM neuronal cell adhesion molecule MDCK Madin-Darby canine kidney (cells) NCBI National Center for Biotechnology MDM multiply deficient medium Information 2-ME 2-mercaptoethanol NCI National Cancer Institute MEF mouse embryo fibroblasts NCS newborn calf serum MEM minimal essential medium ND not determined MEMPFA MOPS/sodium chloride/ NDV Newcastle Disease Virus magnesium sulfate/paraformaldehyde NGF nerve growth factor (buffer) NLM National Library of Medicine MES 2-(N-morpholino)ethanesulfonic acid neo neomycin gene (selectable marker) MHz megahertz NEPHGE nonequilibrium pH gradient MIPS Martinsried Institute for Protein electrophoresis Sequences Neu5Ac N-acetyl-D-neuraminic acid miRNA microRNA Neu5Gc N-glycolyl-D-neuraminic acid Mls minor lymphocyte stimulating NHS N-hydroxysuccinimide determinant NICHD National Institute of Child Health αMM α-methyl-D-mannoside and Human Development MMC mixed-mode chromatography NIH National Institutes of Health Standard mmCIF macromolecular crystallographic NK natural killer (cells) Measurements, information file NLM National Library of Medicine Data, and Abbreviations A.1A.5

Current Protocols in Molecular Biology Supplement 70 NMR nuclear magnetic resonance PIPES piperazine-N,N-bis(2-ethane NP-40 Nonidet P-40 (detergent) sulfonic acid) NPC normal-phase chromatography PITC phenylisothiocyanate NPP nitrophenyl phosphate PKC protein kinase C nr nonredundant PLAP placental alkaline phosphatase nt nucleotide PMF peptide mass fingerprint NTA nitrilotriacetic acid PMS pregnant mare’s serum NTA-SAM nitrilotriacetic acid PMSF phenylmethylsulfonyl fluoride self-assembled monolayer PMT photomultiplier tube NTP nucleoside triphosphate PNA peanut agglutinin OCT optimal cutting temperature (medium) PNGase-F Peptide:N-Glycosidase F o.d. outer diameter PNP purine nucleoside kinase OD260 optical density at 260 nm pNP p-nitrophenol OGT O-GlcNAc transferase PNPP p-nitrophenyl phosphate oligo oligonucleotide, a short, poly(A) polyadenylic acid or polyadenylate single-stranded DNA or RNA. poly(A)+ polyadenylated (mRNA) oligo(dT) oligodeoxythymidylic acid poly(A)− nonpolyadenylated (mRNA) OMIM Online Mendelian Inheritance in poly(dA-dT) poly(deoxyadenylic Man acid-deoxythymidylic acid) ONPG o-nitrophenyl-β-D-galactosidase poly(U) polyuridylic acid or polyuridylate ORC origin recognition complex PP1 protein phosphatase 1 ORF open reading frame PP2A protein phosphatase 2A ori origin of replication PP2B protein phosphatase 2B PAC P1-derived artificial chromosome; PPase thermostable pyrophosphatase phenoxyacetyl ppm parts per million PAD pulsed amperometric detection PPO 2,5-diphenyloxazole PAGE polyacrylamide gel electrophoresis PR prosthetic-group removing PAH polyaromatic hydrocarbons PRF Protein Research Foundation PAP peroxidase-anti-peroxidase (reaction) PRINS primed in situ (labeling) par partition loci on plasmid DNA Pristane 2,6,10,14-tetramethylpentadecane PB phosphate buffer ProTherm Thermodynamic Database for PBMC peripheral blood mononuclear cells Proteins and Mutants PBS phosphate-buffered saline PS copoly(styrene-1%-divinylbenzene) PCD programmed cell death PSF point-spread function PCMB parachloromercuric benzoate PSI-BLAST Position-Specific Iterated PCR polymerase chain reaction BLAST PDB Protein Data Bank Pth 3-phenyl-2-thiohydrantoin PDD Protein Disease Database PTP protein tyrosine phosphatase PDMP 1-phenyl-2-decanoylamino-3- Pu purine morpholino-1-propanol PVA polyvinyl alcohol PE phycoerythrin PVC polyvinyl chloride PEEK polyethylether ketone PVDF polyvinylidene difluoride PEG polyethylene glycol PVP polyvinylpyrrolidone PEGA polyethylene glycol polyacrylamide Py pyrimidine PEI polyethylenimine PyAOP 7-azabenzotriazol-1-yl- PEO polyethylene oxide oxytris(pyrrolidino)phosphonium PFA paraformaldehyde hexafluorophosphate PFGE pulsed-field gel electrophoresis PyBOP benzotriazolyl-N-oxy- pfu plaque-forming units tris(pyrrolidino)phosphonium PG proteoglycan hexafluorophosphate PGK phosphoglycerate kinase rA riboadenylate pI isoelectric point RACE rapid amplification of cDNA ends PI phosphatidylinositol; propidium iodide RAS Ribi adjuvant system PI-PLC phosphatidylinositol-specific RBC red blood cell phospholipase C RBE relative biological effectiveness PIR Protein Information Resource RBS ribosome-binding site Common PITC phenylisothiocyanate RCA I Ricinus communis agglutinin Abbreviations A.1A.6

Supplement 70 Current Protocols in Molecular Biology RCF relative centrifugal force SSCP single-stranded conformation RDA representational difference analysis polymorphism RE restriction endonuclease SSAV simian sarcoma-associated virus Red-gal 6-chloro-3-indoyl-β-D- sss sheared salmon sperm galactopyranoside STBS suspension Tris-buffered saline RF replicative form STO SIM mouse embryo fibroblasts RFLP restriction-fragment-length resistant to thioguanine and oubain polymorphisms STS sequence tagged site RIA radioimmunoassay STZ streptozotocin RIPA RadioImmunoPrecipitation Assay Sv Sievert (unit for radiation dosage) RMDD restriction-mediated differential T thymine or thymidine; one-letter code for display threonine RNA ribonucleic acid TAE Tris/acetate (buffer) RNAi RNA interference Taq Thermus aquaticus DNA (polymerase) RNase ribonuclease TAU Triton/acetic acid/urea RP reversed phase (HPLC) TBE Tris/borate (buffer) RRE Rev-responsive element TBP TATA box-binding protein rRNA ribosomal ribonucleic acid TBS Tris-buffered saline RT reverse transcriptase TBT TATA-binding protein RT-PCR reverse transcription/polymerase TBTU O-benzotriazol-1-yl-N,N,N,N- chain reaction tetramethyluronium tetrafluoroborate RU resonance unit TCA trichloracetic acid RXR retinoid X receptor TCEP tris(2-carboxyethyl)phosphine SAGE serial analysis of gene expression TCP Transmission Control Protocol SAM S-adenosylmethionine TCR T cell receptor Sarkosyl N-lauroylsarcosine TDM trehalose dimycolate SAX strong anion exchange TdT terminal deoxynucleotidyl transferase SBH sequencing by hybridization TE Tris/EDTA (buffer) SCOP Structural Classification of Proteins TEA triethanolamine acetate SCX strong cation exchange TEAE triethylaminoethyl SD standard deviation TEMED N,N,N,N- SDS sodium dodecyl sulfate tetramethylethylenediamine SE size exclusion (chromatography) TEN NaCl in TE buffer SEAP secreted alkaline phosphatase TES N-tris(hydroxymethyl)methyl-2- SEC size-exclusion chromatography aminoethanesulfonic acid SED standard enzyme diluent TFA trifluoroacetic acid SELDI surface-enhanced laser TFFH tetramethylfluoroformamidinium desorption/ionization hexafluorophosphate SFFV spleen focus-forming virus TFMSA trifluoromethanesulfonic acid SHMT serine hydroxymethyl synthetase TGN trans-Golgi network ShrAP shrimp alkaline phosphatase THF tetrahydrofuran Sia sialic acid TIFF tagged-image file format siRNA short interfering RNA TIR total internal reflection SM suspension medium TK thymidine kinase SNA Sambucus nigra agglutinin TLC thin-layer chromatography SNP single-nucleotide polymorphism TLD thermoluminescent dosimeter SPF specific pathogen free Tm melting (or midpoint) temperature; SPPS solid-phase peptide synthesis thermal denaturation SPR surface plasmon resonance TMAC tetramethylammonium chloride SPRI solid-phase reversible immobilization TMB 3,3,5,5-tetramethylbenzidine SPW surface plasma or plasmon wave TMP trimethylphosphate; thymidine SRBC sheep red blood cells monophosphate SREBP steroid response element binding TMV Tobacco Mosaic Virus protein TONPG orthonitrophenyl-β-D- ss single stranded thiogalactoside Standard SSB single-stranded DNA-binding protein TPCK N-p-tosyl-L-phenylalanine Measurements, SSC sodium chloride/sodium citrate (buffer); chloromethyl ketone Data, and Abbreviations side (light) scatter (in flow cytometry) TPF tiling path format A.1A.7

Current Protocols in Molecular Biology Supplement 70 Tris tris(hydroxymethyl)aminomethane VRC vanadyl-ribonucleoside complex TRITC tetramethylrhodamine V0 void volume isothiocyanate vol/vol; v/v volume/volume tRNA transfer ribonucleic acid VSG variant surface glycoprotein TS thymidylate synthetase VSV vesicular stomatitis virus TSA Tris/saline/azide (buffer) WAIS Wide Area Information Service TTP thymidine 5-triphosphate WAX weak anion exchange TUNEL TdT-mediated dUTP biotin WCX weak cation exchange nick-end labeling WGA wheat germ agglutinin U unit; uracil or uridine WR Western Reserve strain (vaccinia) UAS upstream activating sequence WT wild-type UDG uracil DNA glycosylase wt/vol; w/v weight/volume UDP uridine 5-diphosphate WWW World Wide Web UDP-Gal uridine diphospho-D-galactose XBE Rex-binding element UF ultrafiltration Xgal 5-bromo-4-chloro-3-indolyl-β-D- UMP uridine 5-monophosphate galactoside UPHS U.S. Public Health Service XGPRT xanthine-guanine phosphoribosyl URL uniform resource locator transferase USDA United States Department of Xyl xylose Agriculture Xyl-A 9-β-D-xylofuranosyl adenine UTP uridine 5-triphosphate YAC yeast artificial chromosome UTR untranslated leader region YCp yeast centromeric plasmid UV ultraviolet YEp yeast episomal plasmid UWGCG University of Wisconsin Genetics YIp yeast integrating plasmid Computer Group YNB-AA/AS yeast nitrogen base without VA F viral-antibody free amino acids or ammonium sulfate VAST Vector Alignment Search Tool YPD yeast/peptone/dextrose (medium) Vent Thermococcus litoralis DNA YRp yeast replicating plasmid (polymerase)

Common Abbreviations A.1A.8

Supplement 70 Current Protocols in Molecular Biology Useful Measurements and Data APPENDIX 1B

Figure A.1B.1 A physical chemist’s view of the cell. The data in this figure were assembled from The Molecular Biology of the Cell by Alberts, 1994, and represent the approximate concentrations of a variety of intracellular components.

INORGANIC IONS (ca. 1% w/w) Inside: Outside (eukaryotes): + Na 5 -15 mM 145 mM K+ 140 mM 5 mM + Mg2 30 mM 1-2 mM + Ca2 1-2 mM 2.5 -5 mM DIAMETER (although <10–7 M is free) Cl– 4 mM 110 mM Prokaryotes: pH = 7.4 <1-10 µm Eukaryotes: 10-120 µm

SMALL MOLECULES

70% (w/w) water BIG MOLECULES 3% (w/w) sugars (monomers) 2% (w/w) lipids 22% (w/w) proteins, nucleic 0.4% (w/w) amino acids (monomers) acids, polysaccharides 0.4% (w/w) nucleotides (monomers)

Table A.1B.1 Conversion Factors

Molecular weight (ave.) of DNA base pair: 649 Da 1 kb DNA: 333 amino acids of coding capacity Molecular weight (ave.) of amino acid: 110 Da ≈ 36,000 Da 1 µg/ml DNA: 3.08 µM phosphate 6.5 × 105 Da of double-stranded DNA (sodium salt) 1 µg/ml of 1 kb DNA: 3.08 nM 5′ ends 3.3 × 105 Da of single-stranded DNA (sodium salt) 1 µmol pBR322 (4363 bp): 2.83 g 3.4 × 105 Da of single-stranded RNA (sodium salt) 1 pmol linear pBR322 5′ ends: 1.4 µg µ ≈ 1 A260 double-stranded DNA: 50 g/ml 10 kDa protein 91 amino acids µ ≈ 1 A260 single-stranded DNA: 37 g/ml 273 nucleotides

Table A.1B.2 Genome Size of Various Organismsa Base pairs/ Base pairs/ Organism Organism haploid genome haploid genome SV40 5,243 Drosophila melanogaster 1.4 × 108 ΦX174 5,386 Gallus domesticus (chicken) 1.2 × 109 Adenovirus 2 35,937 Mus musculus (mouse) 2.7 × 109 Lambda 48,502 Rattus norvigeticus (rat) 3.0 × 109 Escherichia coli 4.7 × 106 Xenopus laevis 3.1 × 109 Saccharomyces cerevisiae 1.5 × 107 Homo sapiens 3.3 × 109 Dictyostelium discoideum 5.4 × 107 Zea mays 3.9 × 109 Arabidopsis thaliana 7.0 × 107 Nicotiana tabacum 4.8 × 109 Caenorhabditis elegans 8.0 × 107 aGenome size determined either by direct sequence analysis (viruses), electrophoretic analysis (E. coli, S. cerevisiae), or Standard a combination of DNA content per cell and hybridization kinetics. Some data are from Gene Expression 2 by Lewin, 1980. Measurements, Data, and Abbreviations Current Protocols in Molecular Biology (1997) A.1B.1 A.1B.1 Copyright © 1997 by John Wiley & Sons, Inc. Supplement 44 APPENDIX 1C Characteristics of Amino Acids

PHYSICAL PROPERTIES The physical properties of the amino acids determine the structure and function of the proteins in which they are found. Some useful details and relevant physical characteristics of the amino acids can be found in Table A.1C.1. A detailed view of the chemical structures of the amino acids, and an explanation of the role these structures play in enzymes, can be found in Figure A.1C.2. The three-dimensional structure of proteins is largely deter- mined by the packing of their hydrophobic cores; the properties of amino acids that govern this packing are their relative hydrophobicities, which are presented in Figure A.1C.3, and their shapes and volumes, which can be assessed by referring to the space-filling models shown in Figure A.1C.4. While these figures can be useful in rationalizing amino acid functionality, it is also important to consider how natural selection views the interchangeability of amino acids, as diagrammed in Figure A.1C.5 (and Table A.1C.1).

Post-translational modifications will change the mass of a protein or peptide; values for some common mass changes are listed in Table A.1C.3. Mass changes due to some post-translational modifications are found in Table A.1C.4.

CODON USAGE (see Table A.1C.2) While the amino acid sequence of a protein is selected for in part because of the physical properties of the amino acids themselves, a second, more subtle selection may also operate at the level of the genetic code to determine the sequence of both protein and gene. The genetic code is degenerate. Any of several codons can represent a single amino acid (up to six, in the cases of Arg, Leu, and Ser). However, the frequency with which such synonymous codons are used is not equivalent. Considerations of the bias in codon usage may be relevant to design of synthetic genes (UNIT 8.2B), strategies for overexpression of foreign proteins, particularly in E. coli (see UNIT 16.1), and minimizing degeneracy of oligonucleotide probes and primers (UNIT 6.4 and UNIT 15.1). The reasons for deviation from random usage seem to differ from organism to organism. For E. coli and other microorganisms, it is thought that the codons used more frequently correspond to abundant tRNAs, while the underrepresented codons are those associated with less abundant tRNAs. Since this bias seems particularly strong for genes encoding highly expressed proteins, it is thought to be related to maximizing translation efficiency. In higher organisms, the bias in codon usage may be more closely associated with selection pressures acting at the level of DNA. Mammalian genomes, in particular, show quite significant reductions in the frequency of the dinucleotide CpG, which is a site for methylation. In mammalian genes, codons containing this sequence can be quite strongly underrepresented. It is worth emphasizing that the bias against particular codons is not absolute. While there may be strong trends within a particular organism, individual genes (particularly those expressed at low levels) may deviate substantially (see Sharp et al., 1988). Interestingly, codon usage is quite similar within the broad groups presented (see Wada et al., 1990, for a comparison of mammals).

Characteristics of Amino Acids A.1C.1 Contributed by Andrew Ellington and J. Michael Cherry Current Protocols in Molecular Biology (1997) A.1C.1-A.1C.12 Copyright © 1997 by John Wiley & Sons, Inc. Supplement 44 Table A.1C.1 Physical Characteristics of the Amino Acids

3-letter 1-letter Mol. wt. Accessible Hydro- Relative Surface Amino acid code code (g/mol) surface areaa phobicityb mutabilityc probabilityd Alanine Ala A 89.1 115 −0.40 100 62 Arginine Arg R 174.2 225 −0.59 65 99 Asparagine Asn N 132.1 160 −0.92 134 88 Aspartate Asp D 133.1 150 −1.31 106 85 Cysteine Cys C 121.2 135 0.17 20 55 Glutamate Glu E 147.1 190 −1.22 102 82 Glutamine Gln Q 146.2 180 −0.91 93 93 Glycine Gly G 75.1 75 −0.67 49 64 Histidine His H 155.2 195 −0.64 66 83 Isoleucine Ile I 131.2 175 1.25 96 40 Leucine Leu L 131.2 170 1.22 40 55 Lysine Lys K 146.2 200 −0.67 56 97 Methionine Met M 149.2 185 1.02 94 60 Phenylalanine Phe F 165.2 210 1.92 41 50 Proline Pro P 115.1 145 −0.49 56 82 Serine Ser S 105.1 115 −0.55 120 78 Threonine Thr T 119.1 140 −0.28 97 77 Tryptophan Trp W 204.2 255 0.50 18 73 Tyrosine Tyr Y 181.2 230 1.67 41 85 Valine Val V 117.1 155 0.91 74 46 aAccessible surface area is in Å2 and is for the amino acid as part of a polypeptide backbone (Chothia, 1976). bHydrophobicity is in arbitrary units and is based on the OMH scale of Sweet and Eisenberg (1983), which emphasizes the ability of amino acids to replace one another during the course of evolution. cRelative mutability is also in arbitrary units (with alanine set to 100) and represents the probability that an amino acid will mutate within a given time. Thus, as two closely related proteins diverge, a given tryptophan residue is only 18% as likely as a given alanine residue to mutate (Dayhoff et al., 1978). dSurface probability is the likelihood that 5% or more of the surface area of an amino acid will be exposed to the solution surrounding a protein (Chothia, 1976). Thus, while some portion of almost all the arginines will help make up the surface of a protein, less than half of the valines will be exposed to solution. To understand in more detail how amino acids are buried, see Rose et al. (1985); for example, although tyrosine is often found exposed to the surface of a protein, a substantial proportion of its surface area is typically buried.

e u G Gly h Figure A.1C.1 The genetic code. e lu P L Names of amino acids and chain A Ser s C AG TC A G termination codons are on the p G T T A CA C G Tyr periphery of the circle. The first base Ala T re G T T och r of the codon is identified in the A G C mbe C A C A a center ring; the second base of the G T A Cys codon is in the middle ring; and the G C T Val A C UGA third base(s) of the codon is in the C T G T G A T G Trp outer ring of the circle. Arg G T A G T C C AC A Leu Ser T A C G G T Lys A C C C A A T T G G Pro Asn G A T C A C T G G His Standard Thr A C A C T Measurements, TG Gln IIe Arg Data, and Met Abbreviations A.1C.2

Current Protocols in Molecular Biology Supplement 33 Table A.1C.2 Percentage of Codon Synonomous Usage and Frequency of Codon Occurrence in Various Organisms (see description below)

Supplement 33 Supplement Mammal Other vert. Dicot Monocot Invertebrate Yeast Chloroplast Yeast mito. Gram neg. Gram pos. AA Codon

A.1C.3 %freq.%freq.%freq.%freq.%freq.%freq.%freq.%freq.%freq.%freq. Gly GGG 22.8 16.6 20.7 14.8 11.6 9.4 25.0 20.5 8.4 6.0 9.0 5.2 15.3 13.8 6.0 3.8 12.3 9.4 13.7 9.2 Gly GGA 25.5 18.5 29.2 20.9 37.9 30.6 21.2 17.4 32.4 23.3 15.0 8.5 38.1 34.4 21.0 12.8 9.2 7.0 30.5 20.5 Gly GGU 17.6 12.8 21.3 15.2 35.6 28.8 15.2 12.4 21.4 15.6 61.0 34.9 35.6 32.2 68.0 40.9 32.9 25.1 27.2 18.2 Gly GGC 34.1 24.7 28.9 20.7 14.9 12.1 38.6 31.6 37.8 25.7 15.0 8.9 11.0 9.9 4.0 2.2 45.6 34.9 28.5 19.1 Glu GAG 60.1 40.4 55.9 42.3 48.9 29.3 72.5 31.9 66.0 38.4 26.0 17.0 23.7 14.4 20.0 7.2 34.5 21.2 26.6 19.3 Glu GAA 39.9 26.8 44.1 33.4 51.1 30.6 27.5 12.1 34.0 20.7 74.0 49.4 76.3 46.5 80.0 28.9 65.5 40.2 73.4 53.2 Asp GAU 42.6 20.9 50.9 26.2 60.1 26.4 29.7 10.2 53.0 25.2 62.0 37.2 75.4 28.1 72.0 26.7 55.2 30.9 67.6 38.8 Asp GAC 57.4 28.1 49.1 25.3 39.9 17.5 70.3 24.1 47.0 22.4 38.0 22.6 24.6 9.2 28.0 10.4 44.8 25.1 32.4 18.6 Val GUG 48.1 29.5 42.0 25.7 28.6 19.3 38.0 23.4 41.6 22.8 15.0 9.6 15.2 9.9 8.0 4.6 34.2 23.8 21.9 13.7 Val GUA 9.9 6.1 13.1 8.0 14.0 9.4 9.5 5.8 10.6 6.1 16.0 9.7 40.6 26.4 46.0 27.7 16.0 11.1 25.8 16.1 Val GUU 16.4 10.0 22.0 13.5 40.0 26.9 18.8 11.6 21.1 12.0 44.0 27.4 34.3 22.3 37.0 22.1 25.9 18.0 32.2 20.1 Val GUC 25.6 15.7 22.9 14.0 17.4 11.7 33.7 20.8 26.8 15.3 24.0 15.1 9.9 6.4 9.0 5.2 23.9 16.6 20.0 12.5 Ala GCG 9.9 7.0 8.8 6.5 6.6 4.8 25.3 21.7 18.1 13.9 8.0 5.0 10.1 7.7 5.0 3.2 34.3 33.3 21.4 14.7 Ala GCA 21.0 14.9 26.7 19.6 27.0 19.4 18.0 15.5 18.2 14.7 23.0 15.0 26.4 20.2 36.0 21.7 19.3 18.7 35.7 24.6 Ala GCU 28.8 20.4 33.2 24.4 44.6 32.0 21.8 18.7 21.4 17.3 44.0 28.5 48.5 37.1 51.0 30.7 17.2 16.7 27.6 19.0 Ala GCC 40.2 28.5 31.3 23.0 21.8 15.6 34.9 30.0 42.4 33.0 25.0 16.0 15.0 11.5 8.0 4.8 29.2 28.3 15.4 10.6 Arg AGG 21.5 11.8 21.4 12.0 25.1 11.9 24.4 12.7 14.1 9.4 17.0 7.3 9.3 6.3 11.0 3.2 3.2 1.9 9.9 3.8 Arg AGA 21.0 11.5 23.6 13.2 32.6 15.4 13.0 6.8 14.6 9.8 54.0 23.6 30.1 20.2 70.0 20.3 4.3 2.5 33.1 12.6 Ser AGU 13.4 10.0 13.2 9.6 16.4 12.8 7.7 5.5 12.4 10.4 14.0 11.1 19.7 12.8 15.0 11.0 11.8 7.0 14.6 9.9 Ser AGC 24.8 18.6 25.8 18.7 17.3 13.5 22.1 15.6 22.4 18.4 9.0 7.4 10.1 6.6 5.0 3.4 28.0 16.6 20.5 13.9 Lys AAG 62.4 36.1 58.4 38.0 57.7 33.0 80.5 28.8 70.6 37.0 49.0 35.3 28.7 14.6 25.0 13.6 29.5 14.2 24.2 19.8 Lys AAA 37.6 21.7 41.6 27.1 42.3 24.2 19.5 7.0 29.4 15.5 51.0 36.8 71.3 36.1 75.0 41.7 70.5 33.9 75.8 61.8 Asn AAU 41.4 15.5 43.8 17.0 45.9 20.7 25.9 7.6 44.1 18.9 54.0 31.0 66.5 25.2 86.0 66.2 37.8 15.5 58.2 31.3 Asn AAC 58.6 22.0 56.2 21.8 54.1 24.4 74.1 21.9 55.9 24.3 46.0 25.9 33.5 12.7 14.0 10.8 62.2 25.6 41.8 22.5 Met AUG 100 22.6 100 23.4 100 22.3 100 20.6 100 23.2 100 21.3 100 24.0 65.0 28.7 100 25.2 100 23.4 Ile AUA 13.1 5.9 16.8 7.8 18.9 9.9 13.6 5.5 15.6 7.1 20.0 12.1 24.5 18.9 35.0 15.4 7.3 4.1 18.7 12.4 Ile AUU 33.3 14.9 35.8 16.7 46.2 24.2 27.5 11.1 33.8 15.4 50.0 30.7 51.7 39.9 86.0 67.8 42.6 23.9 51.5 34.2 Ile AUC 53.6 24.0 47.4 22.0 34.9 18.2 58.9 23.8 50.6 23.5 30.0 18.4 23.8 18.4 14.0 11.2 50.1 28.0 29.8 19.8 Thr ACG 11.8 6.6 11.3 6.3 8.5 4.7 21.1 10.7 24.7 13.8 12.0 6.8 9.0 4.8 6.0 2.8 24.4 13.4 20.3 11.8 Thr ACA 26.4 14.8 30.3 17.1 29.3 16.1 18.9 9.6 21.8 12.7 26.0 15.4 30.1 16.0 46.0 20.1 11.5 6.3 47.3 27.4 Thr ACU 23.4 13.1 26.0 14.7 35.0 19.3 20.9 10.6 18.0 10.2 38.0 22.5 40.4 21.6 38.0 16.7 18.8 10.3 21.3 12.4 Thr ACC 38.5 21.6 32.4 18.3 27.2 15.0 39.0 19.8 35.5 20.2 25.0 14.5 20.5 10.9 10.0 4.4 45.3 24.9 11.0 6.4 Current Protocols in Molecular Biology Molecular in Protocols Current Trp UGG 100 14.4 100 12.5 100 14.7 100 13.0 100 11.6 100 10.2 100 11.9 39.0 6.8 100 13.3 100 9.7 End UGA 59.6 2.6 58.1 2.9 51.4 3.8 60.3 3.7 56.2 3.9 34.0 0.7 23.1 0.9 61.0 10.6 33.2 1.0 18.9 0.6 Cys UGU 42.7 10.0 39.7 8.7 49.3 8.9 27.0 5.5 31.5 7.3 68.0 7.9 71.4 6.6 87.0 6.6 38.0 4.2 47.8 3.0 Cys UGC 57.3 13.4 60.3 13.2 50.7 9.2 73.0 14.9 68.5 15.1 32.0 3.8 28.6 2.6 13.0 1.0 62.0 6.8 52.2 3.3 End UAG 17.2 0.7 13.8 0.7 18.0 1.3 21.3 1.3 15.3 1.0 17.0 0.4 15.4 0.6 7.0 0.2 9.7 0.3 15.6 0.5 End UAA 23.2 1.0 28.0 1.4 30.6 2.3 18.4 1.1 28.5 2.0 49.0 1.0 61.5 2.3 93.0 2.6 57.2 1.8 65.6 2.1 Tyr UAU 40.2 11.5 41.6 11.2 46.9 14.4 25.7 7.3 36.9 9.4 50.0 16.3 71.6 23.2 82.0 36.3 50.6 15.2 70.6 26.3 Tyr UAC 59.8 17.2 58.4 15.8 53.1 16.3 74.3 21.1 63.1 16.2 50.0 16.6 28.4 9.2 18.0 8.2 49.4 14.8 29.4 11.0 Leu UUG 12.2 11.4 13.8 12.0 26.1 23.0 15.7 13.0 16.8 13.4 36.0 32.6 21.5 19.7 10.0 13.2 11.6 11.1 15.0 12.9 Leu UUA 5.4 5.0 6.5 5.6 11.1 9.8 4.6 3.8 7.0 5.6 27.0 24.1 31.5 28.8 72.0 92.3 9.4 9.0 29.5 25.4 Phe UUU 40.7 15.3 44.2 14.6 49.5 21.3 30.6 10.1 34.8 11.3 53.0 22.7 59.8 24.0 60.0 34.5 47.5 17.1 68.0 27.3 Phe UUC 59.3 22.3 55.8 18.4 50.5 21.8 69.4 22.9 65.2 21.2 47.0 20.0 40.2 16.2 40.0 23.1 52.5 18.9 32.0 12.8 Current Protocols in Molecular Biology Molecular in Protocols Current Mammal Other vert. Dicot Monocot Invertebrate Yeast Chloroplast Yeast mito. Gram neg. Gram pos. AA Codon %freq.%freq.%freq.%freq.%freq.%freq.%freq.%freq.%freq.%freq.

Ser UCG 5.9 4.4 6.0 4.3 5.8 4.5 17.0 12.0 19.5 15.7 8.0 6.5 7.0 4.5 4.0 3.0 15.4 9.1 8.6 5.8 Ser UCA 14.2 10.6 15.4 11.2 19.7 15.3 16.0 11.3 11.4 9.9 19.0 15.3 16.1 10.5 44.0 31.5 10.7 6.4 22.2 15.0 Ser UCU 18.3 13.8 19.3 14.0 23.6 18.3 15.3 10.8 11.3 9.7 32.0 25.4 27.1 17.6 25.0 17.7 16.1 9.6 23.4 15.9 Ser UCC 23.5 17.6 20.4 14.8 17.2 13.4 21.9 15.5 23.0 18.9 18.0 14.8 20.0 13.0 7.0 4.8 18.0 10.7 10.7 7.3 Arg CGG 18.2 10.0 13.3 7.5 5.0 2.4 16.4 8.6 13.8 8.7 2.0 1.0 6.1 4.1 4.0 1.2 9.6 5.6 10.8 4.1 Arg CGA 10.7 5.8 9.4 5.3 9.5 4.5 9.0 4.7 14.1 9.3 5.0 2.1 20.1 13.5 1.0 0.2 5.6 3.3 10.5 4.0 Arg CGU 9.1 5.0 13.4 7.5 18.8 8.9 11.2 5.8 16.5 10.9 17.0 7.3 25.5 17.2 13.0 3.6 36.9 21.4 19.9 7.6 Arg CGC 19.5 10.7 18.9 10.6 9.0 4.2 26.0 13.6 27.0 17.0 4.0 1.9 8.9 6.0 1.0 0.4 40.4 23.5 15.8 6.0 Gln CAG 73.9 32.7 69.2 30.2 39.7 15.2 40.1 43.4 65.0 32.8 26.0 10.3 26.8 9.9 17.0 4.4 70.2 29.4 36.4 15.3 Gln CAA 26.1 11.6 30.8 13.5 60.3 23.1 59.9 64.8 35.0 18.2 74.0 29.7 73.2 27.1 83.0 21.9 29.8 12.5 63.6 26.7 His CAU 38.8 9.2 43.1 10.0 53.0 12.0 38.2 8.1 41.6 12.1 60.0 12.5 71.5 14.3 83.0 19.5 50.1 11.1 70.4 15.8 His CAC 61.2 14.5 56.9 13.3 47.0 10.7 61.8 13.2 58.4 16.5 40.0 8.3 28.5 5.7 17.0 4.0 49.9 11.1 29.6 6.6 Leu CUG 42.8 40.1 40.3 34.9 11.0 9.6 28.2 23.2 38.1 30.0 9.0 8.5 6.2 5.7 5.0 6.4 53.7 51.5 18.5 15.9 Leu CUA 6.8 6.4 7.2 6.3 9.8 8.6 9.8 8.1 8.6 7.0 13.0 11.8 15.4 14.1 6.0 8.0 3.3 3.1 7.4 6.4 Leu CUU 12.1 11.3 14.8 12.8 25.2 22.2 13.4 11.0 12.4 10.3 11.0 9.6 19.6 17.9 5.0 6.0 9.9 9.5 21.6 18.6 Leu CUC 20.8 19.5 17.4 15.0 16.9 14.8 28.3 23.3 17.2 14.3 4.0 4.0 5.7 5.2 1.0 1.4 12.1 11.6 8.0 6.9 Pro CCG 11.2 6.8 11.0 6.0 8.2 4.6 25.7 20.5 27.3 15.5 9.0 4.1 12.8 6.3 5.0 2.0 54.4 23.7 30.3 10.8 Pro CCA 27.3 16.5 30.4 16.7 41.9 23.6 40.9 32.6 30.1 17.9sativum; 49.0 21.9 23.3 11.4 39.0 15.6 17.1 7.4 28.9 10.3 Pro CCU 28.8 17.4 28.6 15.7 34.1 19.2 15.0 12.0 14.6 8.9 29.0 12.7 43.3 21.1 48.0 19.3 15.2 6.6 33.2 11.9 Pro CCC 32.7 19.7 30.0 16.4 15.9 8.9 18.3 14.6 28.0 16.1 13.0 5.7 20.6 10.0 8.0 3.4 13.3 5.8 7.6 2.7 esculentum;

A discussion of codon randomness and the relevance of this phenomenon in molecular vertebrate: Total of 159, 994 codons from chicken (Gallus sp.; 72.24%) and Xenopus laevis biology experimentation can be found on p. A.1.6. (% Column) Percentage of synonymous (27.76%). Dicot: Total of 71, 408 codons from Arabidopsis thaliana (15.21%), pea (Pisum codon usage. The relative percentage of each member of the set of codons that specify a 13.77%), Petunia sp. (6.61%), lima bean (Phaseolus vulgaris; 11.24%), potato charomyces cerevisiae. particular amino acid. Four sets that are not contiguous in this table are Arg, Ser, Leu, and (Solanum tuberosum; 7.89%), tobacco (Nicotiana tabacum; 9.54%), tomato (Lycopersicon chain terminator. (Freq. Column) Frequencies are expressed as occurrences per thousand 11.63%), and soybean (Glycine max; 24.10%). Monocot: Total of 45, 622 codons codons for the specified organisms. The GenBank nucleic acid database as of April 23, 1990, from barley (Hordeum vulgare; 21.35%), corn (Zea mays 47.70%), rice (Oryza sativa; was used as the source of gene-coding regions. This corresponds to Genbank Release 63 11.60%) and wheat (Triticum aestivum;19.35%). Invertebrate: Total of 151, 794 codons from with the addition of three weekly updates. The feature tables were utilized to automatically Caenorhabditis elegans (9.11%), sea urchin (Strongylocentrotus purpuratus; 6.01%), and extract peptide-coding regions from the database. This automated process discarded se- fruit( fly (Drosophila melanogaster; 84.88%). Yeast: Total of 216, 375 codons from Sac- quences that were less than ten codons in length and contained more than one chain-termi- Chloroplast: Total of 6, 866 codons from Zea mays chloroplast nation codon. The extracted coding sequences were then examined to remove all (52.18%) and Nicotiana tabacum chloroplast (47.82%). Yeast mitochondrion: Total of 4, 986 psuedogenes, nonnuclear (except for the chloroplast and mitochondrial categories), viral, codons from Saccharomyces cerevisiae mitochondria (see note below). Gram negative rearranged, and mutant sequences. The remaining collection of extracted sequences was bacteria: Total of 263, 904 codons from Escherichia coli (70.40%), Klebsiella pneumoniae edited to remove duplicate entries; most significantly, only one example of each class of 3.96%), Neisseria gonorrheae (1.75%), Pseudomonas sp. (10.97%), Rhizobium meliloti immunoglobulin gene was allowed per species. The codon frequencies were tabulated with (2.57%), and Salmonella typhimurium (10.35%). Gram positive bacteria: Total of 38,807 the aid of the CodonFrequency program from the UWGCG package (Genetics Computer codons from Bacillus subtilis (73.45%) and Staphylococcus aureus (26.55%). Group, Madison, WI 53711). A.1C.4 Supplement 33 Supplement NOTE: The genetic code is universal with the exception of mitochondrial DNA. In yeast The total number of codons used for each organism category and the source of the codons mitochondria the AUA and UGA codons that normally specify isoleucine and chain termination are listed below expressed as a percentage of the total: Mammal: Total of 1,237,027 codons are used for methionine and tryptophan, respectively. These exceptions are used when from cow (Bos taurus; 6.43%), hamster (Cricetulus sp. and Mesocricetus sp.; 1.48%), human calculating the yeast mitochondrion percentage of synonymous codon usage. (Homo sapiens; 48.36%), macaque (Macaca sp.; 0.38%), mouse (Mus sp.; 19.52%), rabbit (Oryctolagus sp.; 4.06%), rat (Rattus sp.; 19.17%), and sheep (Ovis sp.; 0.59%). Other A Amino acids with dissociable protons

10.5 acidic Fig. A.1C.2 OH

4.1 3.9 COOH 8.4 13.7 COOH SH OH H H H H H

+ – + – + – + – + – H3N COO H3N COO H3N COO H3N COO H3N COO aspartate glutamate cysteine tyrosine serine

pKa 34 5 6 7 8 910 11 12 13 14 12.5 NH 6.0 10.5 2+ + H + NH3 NH2 N NH NH

H H H

+ – + – + – H3N COO H3N COO H3N COO histidine lysine arginine

basic

Other amino acids with polar side chains

O O NH 2 NH2 OH H H H H3C H + – + – + – H3N COO H3N COO H3N COO asparagine glutamine threonine

Characteristics of Amino Acids A.1C.5

Supplement 33 Current Protocols in Molecular Biology B H Nonpolar H H H3C H amino acids + – N COO– + – H3NCOO H3NCOO H + H glycine proline alanine

H CH N 3 S CH3 H H3C H H + – + – + – H3NCOO H3NCOO H3NCOO

tryptophan valine methionine

CH3 CH3 CH3 H H H3C H H + – + – + – H3NCOO H3NCOO H3NCOO leucine isoleucine phenylalanine

Figure A.1C.2 Line drawings of the amino acids. The can markedly change when these groups are buried in amino acids are roughly divided into three groups: amino proteins. The pKas of the α-amino groups range from 8.7 to acids with dissociable protons (A), other amino acids with 10.7, while the pKas of the α-carboxylates range from 1.8 to polar side chains (A), and nonpolar amino acids (B). These 2.4. groupings are designed to facilitate an understanding of Amino acids with polar side chains (A) can form hydrogen enzymology and the thermodynamics of protein folding. bonds to substrates or to each other. Cysteine, serine, and In this representation, hydrogens are omitted except in tyrosine could also be included in this group, since the showing ionization or stereochemistry. In the case of ar- ionized forms of these amino acids do not generally perform ginine the delocalized positive charge is indicated by structural roles in proteins. In general, these amino acids dashed double bonds. At stereocenters, bold lines indicate (and the amino acids with dissociable protons) will be found a group is coming out of the page toward the viewer, while on the surfaces of proteins. Cysteine is an exception, since hashed lines indicate that the group goes into the page away it is slightly hydrophobic and can often be buried as a from the viewer. disulfide bond. Amino acids with dissociable protons are generally inti- The nonpolar amino acids (B) are often found in the interiors mately involved in the chemistry of enzymes. Acidic and of proteins or in hydrophobic substrate-binding pockets. basic groups can form salt bridges to substrates or to each They interact with one another like jigsaw pieces, forming other. They can also act as proton donors/acceptors in tight-fitting associations that have a density similar to that of mechanisms that rely on acid/base catalysis. The polar side an amino acid crystal. Proline is buried less frequently than chains of some of these amino acids (notably cysteine, might be expected because of its predominance in turns, serine, and histidine) can act as nucleophiles. The pKa which are often found on the periphery of a protein. values for the free amino acids are shown, but these values Standard Measurements, Data, and Abbreviations A.1C.6

Current Protocols in Molecular Biology Supplement 40 3

2

1

0 Hydrophobicity –1 OMH (arbitrary units) Frömmel (kcal/mol) –2

–3 F Y I L M V W C T A P S R H G K Q N D E Amino acid

Figure A.1C.3 Amino acid hydrophobicity. The hydrophobicity of an amino acid is the degree to which it prefers a nonpolar medium, such as ethanol or the interior of a protein, to a polar medium, such as water. In this graph, the more hydrophobic amino acids “sink” below zero, while the more hydrophilic amino acids “float” above the surface. Two scales are used. The Frömmel scale (Frömmel, 1984) represents the free energy of transfer from a hydrophobic medium to water. This value is an intrinsic property of an amino acid, separate from its role in a protein. In contrast, the OMH scale (Sweet and Eisenberg, 1983) is a measure of how likely a given amino acid will be replaced by a different hydrophobic or “buried” amino acid in a protein. In effect, this scale is how evolution views the hydrophobicity of an amino acid. The distinction between physical and evolutionary properties is important. For example, while arginine is definitely a charged, polar amino acid (Sambrook et al., 1989), it can substitute more freely for nonpolar amino acids in the interior of a protein than glutamate (also a charged, polar amino acid) because of its long aliphatic side chain.

Characteristics of Amino Acids A.1C.7

Supplement 40 Current Protocols in Molecular Biology glycine alanine serine cysteine threonine proline

aspartate valine asparagine leucine isoleucine glutamine

methionineglutamate histidine lysine phenylalanine

CN

H

S O arginine tyrosine tryptophan

Figure A.1C.4 Space-filling representations of the amino acids. The amino acids are arranged in order of size. The conformations shown maximize the two-dimensional area but are not necessarily the most stable geometries.

Standard Measurements, Data, and Abbreviations A.1C.8

Current Protocols in Molecular Biology Supplement 38 W

R K

H

Q N

E D

P C

A S

G T

V I

L

M FY

Figure A.1C.5 Mutational pathways for amino acids. In this diagram, amino acids are parsed into sets based on their ability to replace one another during the evolution of closely related proteins. Dark arrows show the most frequent mutational events for each of the twenty amino acids. For example, tryptophan most frequently mutates to arginine, while arginine and lysine most frequently replace one another. Dotted arrows represent the most frequent replacements between sets of otherwise mutationally related amino acids. Thus, while lysine mutates most frequently to arginine within the [arginine, lysine, tryptophan] set, the most likely event that will occur outside of this set is mutation to asparagine.

Characteristics of Amino Acids A.1C.9

Supplement 38 Current Protocols in Molecular Biology Table A.1C.3 Compositions and Masses of the Twenty Commonly Occurring Amino Acid Residuesa,b

Monoisotopic Average pK of ionizing Occurrence in Name Composition a mass mass side chainc proteins (%)

Alanine (Ala, A) C3H5NO 71.03711 71.0788 — 8.3

Arginine (Arg, R) C6H12N4O 156.10111 156.1876 ~11.5–12.5 (12) 5.7

Asparagine (Asn, N) C4H6N2O2 114.04293 114.1039 — 4.4

Aspartic acid (Asp, D) C4H5NO3 115.02694 115.0886 3.9–4.5 (4) 5.3

Cysteine (Cys, C) C3H5NOS103.00919 103.1448 8.3–9.5 (9) 1.7

Glutamic acid (Glu, E) C5H7NO3 129.04259 129.1155 4.3–4.5 (4.5) 6.2

Glutamine (Gln, Q) C5H8N2O2 128.05858 128.1308 — 4.0

Glycine (Gly, G) C2H3NO 57.02146 57.0520 — 7.2

Histidine (His, H) C6H7N3O 137.05891 137.1412 6.0–7.0 (6.3) 2.2

Isoleucine (Ile, I) C6H11NO 113.08406 113.1595 — 5.2

Leucine (Leu, L) C6H11NO 113.08406 113.1595 — 9.0

Lysine (Lys, K) C6H12N2O 128.09496 128.1742 10.4–11.1 (10.4) 5.7

Methionine (Met, M) C5H9NOS131.04049 131.1986 — 2.4

Phenylalanine (Phe, F) C9H9NO 147.06841 147.1766 — 3.9

Proline (Pro, P) C5H7NO 97.05276 97.1167 — 5.1

Serine (Ser, S) C3H5NO2 87.03203 87.0782 — 6.9

Threonine (Thr, T) C4H7NO2 101.04768 101.1051 — 5.8

Tryptophan (Trp, W) C11H10N2O 186.07931 186.2133 — 1.3

Tyrosine (Tyr, Y) C9H9NO2 163.06333 163.1760 9.7–10.1 (10.0) 3.2

Valine (Val, V) C5H9NO 99.06841 99.1326 — 6.6 aFor corresponding structures, see Figure A.1C.2 and Figure A.1C.4. bThe molecular mass of a normally terminated and unmodified peptide or protein may be calculated by summing the masses of the appropriate amino acid residues and adding the masses of H and OH for the N and C termini, respectively. In cases where cysteines are linked to form disulfide bridges, the mass of two hydrogen atoms should be subtracted for each disulfide bridge in the molecule. Specifically, monoisotopic masses were calculated using the atomic masses of the most abundant isotope of the elements: C = 12.0000000, H = 1.0078250, N = 14.0030740, O = 15.9949146, and S = 31.9720718. Average masses were calculated using the atomic weights of the elements: C = 12.011, H = 1.00794, N = 14.00674, O = 15.9994, and S = 32.066. cThese values are included for anyone wishing to make approximate isoelectric point determinations based on protein composition. α α Values for the terminal residues depend on the identity of the residue: -amino, pKa 6.8–8.2 (8.0); -carboxyl, pKa 3.2–4.3 (3.6). Values in parentheses are based on those given by Matthew et al. (1978) and provide a good starting point for determinations.

Standard Measurements, Data, and Abbreviations A.1C.10

Current Protocols in Molecular Biology Supplement 38 Table A.1C.4 Mass Changes Due to Some Post-Translational Modifications of Peptides and Proteinsa

Monoisotopic Average Modificationb mass change mass change Common modifications Pyroglutamic acid formation from Gln −17.0265 −17.0306 Disulfide bond (cystine) formation −2.0157 −2.0159 C-terminal amide formation from Gly −0.9840 −0.9847 Deamidation of Asn and Gln −0.9840 −0.9847 Methylation 14.0157 14.0269 Hydroxylation 15.9949 15.9994 Oxidation of Met 15.9949 15.9994 Proteolysis of a single peptide bond 18.0106 18.0153 Formylation 27.9949 28.0104 Acetylation 42.0106 42.0373 Carboxylation of Asp and Glu 43.9898 44.0098 Phosphorylation 79.9663 79.9799 Sulfation 79.9568 80.0642 Cysteinylation 119.0041 119.1442 Glycosylation with pentoses (Ara, Rib, Xyl) 132.0423 132.1161 Glycosylation with deoxyhexoses (Fuc, Rha) 146.0579 146.1430 Glycosylation with hexosamines (GalN, GlcN) 161.0688 161.1577 Glycosylation with hexoses (Fru, Gal, Glc, Man) 162.0528 162.1424 Modification with lipoic acid (amide bond to lysine) 188.0330 188.3147 Glycosylation with N-acetylhexosamines (GalNAc, GlcNAc) 203.0794 203.1950 Farnesylation 204.1878 204.3556 Myristoylation 210.1984 210.3598 Biotinylation (amide bond to lysine) 226.0776 226.2994 Modification with pyridoxal phosphate (Schiff base to lysine) 231.0297 231.1449 Palmitoylation 238.2297 238.4136 Stearoylation 266.2610 266.4674 Geranylgeranlylation 272.2504 272.4741 Glycosylation with N-acetylneuraminic acid (sialic acid, 291.0954 291.2579 NeuAc, NANA, SA) Glutathionylation 305.0682 305.3117 Glycosylation with N-glycolylneuraminic acid (NeuGe) 307.0903 307.2573 5′-Adenosylation 329.0525 329.2091 Modification with 4′-phosphopantetheine 339.0780 339.3294 ADP-ribosylation (from NAD) 541.0611 541.3052 Adventitious modifications Acrylamide 71.0371 71.0788 Glutathione 304.0712 304.3038 2-Mercaptoethanol 75.9983 76.1192 aTo obtain the molecular mass of a modified peptide or protein, the appropriate mass changes should be algebraically added to the molecular mass calculated for the unmodified molecule. bA more extensive list of modifications is available from the Delta mass site at http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRDocs/MassSpec/deltamassV2.html. Characteristics of Amino Acids A.1C.11

Supplement 38 Current Protocols in Molecular Biology LITERATURE CITED Chothia, C. 1976. The nature of the accessible and buried surfaces in proteins. J. Mol. Biol. 105:1-14. Dayhoff, M.O., Schwartz, R.M., and Orcutt, B.C. 1978. A model of evolutionary change in proteins. In Atlas of Protein Sequence and Structure (M. Dayhoff, ed.) Vol. 5, pp. 345-352. National Biomedical Research Foundation, Washington, D.C. Frömmel, C. 1984. The apolar surface area of amino acids and its empirical correlation with hydrophobic free energy. J. Theor. Biol. 111:247-260. Rose, G.D., Geselowitz, A.R., Lesser, G.J., Lee, R.H., and Zehfus, M.H. 1985. Hydrophobicity of amino acid residues in globular proteins. Science 229:834-838. Matthew, J.B., Friend, S.H., Botelho, L.H., Lehman, L.D., Hanania, G.I.H., and Gurd, F.R.N. 1979. Biochem. Biophys. Res. Commun. 81:416-421. Sambrook, J., Fritsch, E.F., and Maniatis, T.M. (eds.). 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York. Sharp, P.M., Cowe, E., Higgins, D.G., Shields, D.C., Wolfe, K.H., and Wright, F. 1988. Codon usage patterns in E. coli, B. subtilis, S. cerevisiae, S. pombe, D. melanogaster, and H. sapiens: A review of the considerable within-species diversity. Nucl. Acids Res. 16: 8207-8211. Sweet, R.M. and Eisenberg, D. 1983. Correlation of sequence hydrophobicities measures similarity in three-dimensional protein structure. J. Mol. Biol. 171:479-488. Wada, K.-N., Aota, S.-I., Tsuchiya, R., Ishibashi, F., Gojobori, T., and Ikemura, T. 1990. Codon usage tabulated from the GenBank genetic sequence data. Nucl. Acids Res. 18 (Suppl.):2367-2411.

Contributed by Andrew Ellington and J. Michael Cherry (codon usage) Massachusetts General Hospital Boston, Massachusetts

Standard Measurements, Data, and Abbreviations A.1C.12

Current Protocols in Molecular Biology Supplement 38 Characteristics of Nucleic Acids APPENDIX 1D Nucleic acids have traditionally been regarded solely as informational macromolecules with limited secondary structural features, but recent discoveries have vastly expanded the repertoire of polynucleotide structure and function. While this manual has in general concentrated on how to manipulate DNA and RNA, this section details their structural features. In addition, some experimentally useful properties of the mononucleotide building blocks are listed in Table A.1D.1. The chemical structures of the mononu- cleotides can be seen in Figure A.1D.1, and aspects of nucleotide stereochemistry that are important to an understanding of base pairing and secondary structure can be found in Figure A.1D.2. Although Watson-Crick pairings play a critical role in defining nucleic acid secondary structures, a wide variety of alternative base pairings can be important in higher order conformations; some of these are detailed in Figure A.1D.3. Finally, even given only Watson-Crick-style pairings, secondary structures with significantly different features can be formed. Figure A.1D.4 overviews the differences between A-, B-, and Z-form helices.

Table A.1D.1 Physical Characteristics of the Nucleotidesa

b Mol. wt. λmax λmin εmax TLC mobility Nucleotide − − A280/A260 (g/mol) (nm) (nm) (mM 1 cm 1) ABC ATP 507.2 259 227 15.4 0.15 0 6 34 ADP 427.2 259 227 15.4 0.16 0 26 54 AMP 347.2 259 227 15.4 0.16 11 52 65 Adenosinec 267.2 260 227 14.9 0.14 — — — dATPd 491.2 259 226 15.4 0.15 0 — 35 dAMPd 331.2 259 226 15.2 0.15 11 52 — dA 251.2 260 225 15.2 0.15 — — — CTP 483.2 271 249 9.0 0.97 0 11 41 CDP 403.2 271 249 9.1 0.98 0 33 64 CMP 323.2 271 249 9.1 0.98 15 64 75 Cytidine 243.2 271 250 9.1 0.93 — — — dCTPd 467.2 272 — 9.1 0.98 0 — 43 dCMP 307.2 271 249 9.3 0.99 18 65 — dC 227.2 271 250 9.0 0.97 — — — GTP 523.2 253 223 13.7 0.66 0 5 25 GDP 443.2 253 224 13.7 0.66 0 17 45 GMP 363.2 252 224 13.7 0.66 6 40 51 Guanosinec 283.2 253 223 13.6 0.67 — — — dGTPd 507.2 252 222 13.7 0.66 0 — 26 dGMPd 347.2 253 222 13.7 0.67 6 41 — dG 267.2 254 223 13.0 0.68 — — — UTP 484.2 262 230 10.0 0.38 0 14 49 UDP 404.2 262 230 10.0 0.39 0 41 71 UMP 324.2 262 230 10.0 0.39 20 75 80 Uridine 244.2 262 230 10.1 0.35 — — — TTPd 482.2 267 — 9.6 0.73 0 — 52 TMPd 322.2 267 234 9.6 0.73 24 74 — Thymidined 242.2 267 235 9.7 0.70 — — — a Spectral data are assembled from Fasman (1975) at pH 7.0 except where footnoted otherwise. b TLC mobility is expressed as the percent distance a given spot migrates relative to the solvent front (Rf) in three different TLC systems using 0.5-mm polyethylenimine cellulose plates: “A” is 0.25 M LiCl, “B” is 1.0 M LiCl, and “C” is 1.6 M LiCl. Standard c Spectral measurements taken at pH 6.0. Measurements, d Data, and Spectral data assembled from Dawson et al. (1987). Abbreviations Current Protocols in Molecular Biology (1996) A.1D.1-A.1D.11 A.1D.1 Copyright © 2004 by John Wiley & Sons, Inc. Supplement 66 pKa<1 pKa = 2.4 H H 7N N H H N O 8 56 H O 1 4 N N N H pK = 9.4 5 9 A N pKa = 3.8 G a R 4 R 3 H N H pK = 9.4 3 N 2 N 6 U a H N 2 N H 1 R H O adenosine guanosine uridine

pK <1 H a

H N H O– O– O– – pKa = 6 O O O O 5′ base P P P (04′) γ β α H C N pKa = 4.4 O O O O 4′ 1′ N 3′ 2′ R O HO OH cytidine ribonucleoside 5′ triphosphate pKa = 12.5

HO H3C O base O

H T N H pKa = 10.0 N

dR O HO H

thymidine pKa 14

deoxyribonucleoside

Figure A.1D.1 Line drawings of the nucleotides. The pH values. The pKa values given are for nucleotide mono- chemical structure that predominates at neutral pH is phosphates and were taken from Dawson et al. (1987); a shown. Drawings of the nucleotide bases and their associ- fuller discussion of the chemical basis for these values can ated sugars, either ribose (R) or deoxyribose (dR), are be found in a review by T’so (1974). shown separately. In the representations of ribose (as a The small numbers adjacent to adenosine, uridine, and nucleoside triphosphate) and deoxyribose (as a nucleotide), ribose indicate the nomenclature of the purines, pyrimidi- the bold lines indicate that this portion of the sugar is coming nes, and sugars, respectively. Groups appended to a ring out of the page toward the reader. In this view, the base is have the same numbering as the position to which they are found above the plane of the sugar, while the 3′ hydroxyl linked; thus, the “O6” moiety of guanosine is the carbonyl group is found below the plane of the sugar. oxygen bonded to C6 in the ring. Similarly, “O3′” on ribose The pKa values for all groups are shown; pKas above 7 imply or deoxyribose indicates the oxygen of the hydroxyl group proton dissociation from the pictured structure, while pKas bonded to C3′ in the ring. The α, β, and γ phosphates in a below 7 imply proton association to the pictured structure. nucleoside triphosphate are also indicated. The tautomeric form of a given base may change at different

A.1D.2

Supplement 66 Current Protocols in Molecular Biology H O H O N N N H N H N N H HO HO H N N N H N9 N O O C1′ H

HO OH HO OH

guanosine-anti guanosine-syn

base base ′ C5′ C5 O O ′ C3 C2′

C3′ endo C2′ exo

base C5′ O C3′ C2′

C3′ endo–C2′ exo

Figure A.1D.2 Nucleotide stereochemistry. Depending on different stereochemistries. These are labeled according to the rotation about the bond between C1′ of the sugar and which group is bent out of the plane of the ring, and in which either N1 (for pyrimidines) or N9 (for purines), a nucleotide direction. If a portion of the ring is bent “upward” toward the can be described as either “anti” or “syn.” Because of steric base, this is known as “endo,” while if it is bent “downward” constraints, nucleotides are generally found in the “anti” away from the base, this is known as “exo.” In the figure, configuration, with their Watson-Crick hydrogen bond do- plain lines represent bonds that are within the plane of the nor-acceptors swung outward away from the plane of the sugar, while bold lines indicate that the bond is bent out of sugar ring. However, guanosine is sometimes found in a the plane. Hence, “C3′ endo–C2′ exo” describes a furanose “syn” configuration, both in polynucleotides and in solution. ring in which the 2′ and 3′ carbons have been twisted in In this form, the bulk of the purine ring is positioned directly opposite directions and the bond connecting them crosses over the plane of the sugar. The sugar ring can also adopt the plane of the ring.

Standard Measurements, Data, and Abbreviations A.1D.3

Current Protocols in Molecular Biology Supplement 66 H H

H N N H O H H N O H N H

N N A N H N U H G N H N C H R R N N N N

H O R N H O R

H A:U G:C Watson-Crick pairings

H O H H N H

H H N O H N U H

H N N H N C H N N N G N H O R N R A N O R N R N N H

H H

A:C Wobble pairings G:U

H H

R H H R N N U U

O N O O N O

H H H H

H N N H H N N H

N A N N A N R R N N

H H Hoogsteen Reverse Hoogsteen (parallel chains) (antiparallel chains)

Characteristics of Nucleic Acids A.1D.4

Supplement 66 Current Protocols in Molecular Biology H N O H H H R

O N N N N G N H H N R G N N N

N H N H N N N H O R H H H G-anti:G-syn (antiparallel chains) H

H N H O N H

N H N H N R N N

R O H N

H dR H H C:G—G (as in tRNA Phe) N N N dR H N N H N N H O N

H O N

H N H H N H

N O H "G quartet" N O H N N H N N dR H N N N

H H dR Figure A.1D.3 Base pairing schemas. The chemical struc- acceptor of the purine bases adenosine and guanosine, an tures of the nucleotide bases determine the formation of even wider variety of structures becomes possible, including secondary and tertiary structures in nucleic acids. A wide Hoogsteen base pairs and a G-G pairing in which one of the variety of hydrogen bonding schemas (indicated by dashed guanosine residues assumes a “syn” conformation. Bonds lines) are possible between different bases. Watson-Crick involving N7 of the purine bases allow tertiary structural pairings are perhaps the most widely known and are the interactions to occur in nucleic acids, including triple base basis of the double helical structure of complementary, pairs (such as those found in tRNA) and the recently de- anti-parallel DNA strands. Other base pairs can also be scribed “G quartet” (Sen and Gilbert, 1988). A discussion of accommodated within the double helix, such as “wobble the structural possibilities of base pairing can be found in pairings,” in which the bases are slightly off-center with Saenger’s superlative book, Principles of Nucleic Acid respect to each other. By using the N7 hydrogen bond Structure (1993).

A.1D.5

Current Protocols in Molecular Biology Supplement 33 Figure A.1D.4 Nucleic acid secondary structures. The While both of these helices are right-handed (in terms of structural consequence of the ability of nucleotides to form anthropomorphic referents, if you were to point your thumb Watson-Crick base pairs is nucleic acid double helices. In this along a strand in a 5′ to 3′ manner, the twist of the helix figure, the self-complementary 12-mer CGCGAATTCGCG is would be the same as the curl of your right hand), their shown as both A- and B-form helices. Two representations structural details are very different: B DNA has roughly 10 of the A helix have been shown in order to emphasize the bases per full turn, while A DNA and A RNA have 11 to 12; depth of the major groove. The arrows and brackets in these the major groove of B-form helices is wide and the minor figures are not drawn to scale. groove is narrow, while for A-form helices this is reversed; in

A.1D.6

Supplement 33 Current Protocols in Molecular Biology B-forms the base pairs are located close to the helix axis striking is that found in Z DNA. The Z DNA coil is left- rather (as can be seen in end-on views), while in A-forms the base than right-handed and contains G:C base pairs where the pairs are pushed out away from the long helical axis, leaving G is in the “syn” conformation (shown in the inset). a “hole” in the middle of the polynucleotide coil (if one The uneven progression, or zigzag, of Z DNA can be more imagines DNA as a flat ribbon, then B DNA is twisted from easily seen when the polynucleotide backbone is shown in its ends, while A DNA is coiled on itself). isolation; the inset shows the connectivity between phos- Different helical forms are largely due to differences in sugar phates by 5′ to 3′ vector arrows. Because of its odd shape, stereochemistry. Examples of a 2 ′ endo deoxyribose (found base pairs actually protrude from what would be a cavity in in B DNA) and a 3′ endo deoxyribose (found in A DNA) are A or B DNA; thus, Z DNA has a minor but no major groove. indicated. This diagram is based on the original structure of alternating C:G/G:C base pairs (Wang et al., 1979). While there are a variety of other helical forms, the most A.1D.7

Current Protocols in Molecular Biology Supplement 33 Characteristics of Nucleic Acids A.1D.8

Supplement 33 Current Protocols in Molecular Biology Standard Measurements, Data, and Abbreviations A.1D.9

Current Protocols in Molecular Biology Supplement 33 Characteristics of Nucleic Acids A.1D.10

Supplement 33 Current Protocols in Molecular Biology LITERATURE CITED Dawson, M.C., Elliott, D.C., Elliott, W.H., and Jones, K.M. (eds.). 1987. Data for Biochemical Research, 3rd ed. Clarendon Press, Oxford. Fasman, G. (ed.). 1975. Handbook of Biochemistry and Molecular Biology, Vol. 1: Nucleic Acids, 3rd ed. CRC Press, Boca Raton, Fla. Saenger, W. 1993. Principles of Nucleic Acid Structure. Springer-Verlag, New York. Sen, D. and Gilbert, W. 1988. Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis. Nature 334:364-366. T’so, P.O.P. 1974. Bases, nucleosides, and nucleotides. In Basic Principles in Nucleic Acid Chemistry, Vol. 1 (P.O.P. T’so, ed.) pp. 453-584. Academic Press, San Diego. Wang, A.H., Quigley, G.J., Kolpak, F.J., Crawford, J.L., van Boom, J.H., van der Marel, G., and Rich, A. 1979. Molecular structure of a left-handed double helical DNA fragment at atomic resolution. Nature 282:680-686.

Standard Measurements, Data, and Abbreviations A.1D.11

Current Protocols in Molecular Biology Supplement 33 Radioactivity APPENDIX 1E

ABSTRACT This appendix provides selected properties of radioisotopes commonly used in the molecular biology laboratory. Curr. Protoc. Mol. Biol. 79:A.1E.1-A.1E.5. C 2007 by John Wiley & Sons, Inc. Keywords: radioactivity r isotope r counting efficiency

INTRODUCTION When working with radioactivity, it is important to have a clear understanding of the properties of the radioisotopes and their decay. Table A.1E.1 lists physical characteristics of radioisotopes that are commonly used in the molecular biology laboratory. A discussion of the different types of emissions from radioisotopes can be found in APPENDIX 1F, which provides a thorough discussion of safety considerations for working with radioactivity. For general comparison, Table A.1E.2 illustrates relative shielding capabilities of various materials. The discussion in this unit focuses on aspects of radioactive decay that are important in quantitative analyses.

Table A.1E.1 Physical Characteristics of Commonly Used Radionuclidesa

Approx. specific Decay constant Energy, max Range of Decay Nuclide Emission Half-life activity at 100% (k ) (MeV) emission, max product decay enrichment (Ci/mg)

3 β −1 3 H 12.43 years 0.056 year 0.0186 0.42 cm (air) 9.6 2 He

14 β 14 C 5370 years 0.156 21.8 cm (air) 4.4 mCi/mg 7 N

32 b β −1 32 P 14.3 days 0.0485 day 1.71 610 cm (air) 285 16 S 0.8 cm (water) 0.76 cm (Plexiglas) 33 b β 33 P 25.4 days 0.249 49 cm 156 16 S

35 β −1 35 S 87.4 days 0.079 day 0.167 24.4 cm (air) 43 17 Cl

125 c γ −1 125 I 60 days 0.0116 day 0.027–0.035 0.2 mm (lead) 14.2 52 Te

131 c β 130 I 8.04 days 0.606 165 cm (air) 123 54 Xe γ 0.364 2.4 cm (lead) aTable compiled based on information in Lederer et al. (1967) and Shleien (1987). bRecommended shielding is Plexiglas; half-value layer measurement is 1 cm. cRecommended shielding is lead; half-value layer measurement is 0.02 mm.

Standard Measurements, Data, and Abbreviations

Current Protocols in Molecular Biology A.1E.1-A.1E.5, July 2007 A.1E.1 Published online July 2007 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471142727.mba01es79 Supplement 79 Copyright C 2007 John Wiley & Sons, Inc. Table A.1E.2 Shielding Radioactive Emissiona

β emitters

Thickness (mm) to reduce intensity by 50% Energy Mass (mg)/cm2 to reduce Water Glass Lead Plexiglas (MeV) intensity by 50%

0.1 1.3 0.013 0.005 0.0011 0.0125 1.0 48 0.48 0.192 0.042 0.38 2.0 130 1.3 0.52 0.115 1.1 5.0 400 4.0 1.6 0.35 4.2 γ emitters

Thickness of material (cm) to attenuate a broad beam of γ-rays by a factor of 10 Energy Water Aluminum Iron Lead (MeV)

0.5 54.6 20.3 6.1 1.8 1.0 70.0 24.4 8.2 3.8 2.0 76.0 32.0 11.0 5.9 3.0 89.0 37.0 12.0 6.4 aFrom Dawson et al. (1986). Reprinted with permission.

NOTE: It is important for the researcher to be familiar with applicable regulations and approved procedures for the safe use of radioisotopes. Because the real hazards of low levels of radiation are not known, it is generally assumed that any unavoidable exposure is too much.

RADIOACTIVE DECAY Radioactive decay is an entirely random process. The probability of decay during any particular interval is the same as the probability of decay during any other interval. Starting with N0 radioactive atoms, the number remaining at time t is:

−k t N0 = Nt × e decay

where kdecay is the rate constant of decay in units of inverse time. The half-life (t1/2)is the time it takes for half of the isotope to decay. Each radioisotope has a characteristic value of kdecay and thus a characteristic half-life (Table A.1E.1). The correlation between half-life and remaining radioactivity at any given time is il- lustrated in Figure A.1E.1. Radioactive decay can be calculated from a date when the concentration and specific radioactivity were known using the above equation (i.e., where the fraction remaining is Nt/N0), or can be extrapolated from decay tables for specific isotopes (Table A.1E.3).

Calculations of radioactive decay are straightforward only when each molecule is labeled with a single radioactive isotope, as is usually the case. If a molecule is labeled with several radioactive isotopes, the effective half-life is shorter. If only a fraction of the molecules are labeled with a radioactive isotope, then the decay formula only applies to the labeled portion of the mixture, as the concentration of the unlabeled compound never Radioactivity changes. A.1E.2

Supplement 79 Current Protocols in Molecular Biology Table A.1E.3 Decay factors for calculating the amount of radioactivity present at a given time after a refer- ence date. for example, a vial containing 1.85 MBq (50 µCi) of an 35S-labeled compound on the reference date will have the following activity 33 days later: 1.85 × 0.770 = 1.42 MBq;50× 0.770 = 38.5 µCi

125I Half-Life: 60.0 days 32P Half-Life: 14.3 days Days Hours 0 2 4 6 8 10 12 14 16 18 0 12 24 36 48 60 72 84 0 1.000 0.977 0.955 0.933 0.912 0.891 0.871 0.851 0.831 0.812 0 1.000 0.976 0.953 0.930 0.908 0.886 0.865 0.844

Days 20 0.794 0.776 0.758 0.741 0.724 0.707 0.691 0.675 0.660 0.645 Days 4 0.824 0.804 0.785 0.766 0.748 0.730 0.712 0.695 40 0.630 0.616 0.602 0.588 0.574 0.561 0.548 0.536 0.524 0.512 8 0.679 0.662 0.646 0.631 0.616 0.601 0.587 0.573 60 0.500 0.489 0.477 0.467 0.456 0.445 0.435 0.425 0.416 0.406 12 0.559 0.546 0.533 0.520 0.507 0.495 0.483 0.472 80 0.397 0.388 0.379 0.370 0.362 0.354 0.345 0.338 0.330 0.322 16 0.460 0.449 0.439 0.428 0.418 0.408 0.398 0.389 100 0.315 0.308 0.301 0.294 0.287 0.281 0.274 0.268 0.262 0.256 20 0.379 0.370 0.361 0.353 0.344 0.336 0.328 0.320 120 0.250 0.244 0.239 0.233 0.228 0.223 0.218 0.213 0.208 0.203 24 0.312 0.305 0.298 0.291 0.284 0.277 0.270 0.264 140 0.198 0.194 0.189 0.185 0.181 0.177 0.173 0.169 0.165 0.161 28 0.257 0.251 0.245 0.239 0.234 0.228 0.223 0.217 160 0.157 0.154 0.150 0.147 0.144 0.140 0.137 0.134 0.131 0.128 32 0.212 0.207 0.202 0.197 0.192 0.188 0.183 0.179 180 0.125 0.122 0.119 0.117 0.114 0.111 0.109 0.106 0.104 0.102 36 0.175 0.170 0.166 0.162 0.159 0.155 0.151 0.147 200 0.099 0.097 0.095 0.093 0.090 0.088 0.086 0.084 0.082 0.081 40 0.144 0.140 0.137 0.134 0.131 0.127 0.124 0.121 220 0.079 0.077 0.075 0.073 0.072 0.070 0.069 0.067 0.065 0.064 44 0.119 0.116 0.113 0.110 0.108 0.105 0.102 0.100 240 0.063 0.061 0.060 0.058 0.057 0.056 0.054 0.053 0.052 0.051 48 0.098 0.095 0.093 0.091 0.089 0.086 0.084 0.082 52 0.080 0.078 0.077 0.075 0.073 0.071 0.070 0.068

131I Half-Life: 8.04 days 35S Half-Life: 87.4 days Hours Days 0 6 12 18 24 30 36 42 48 54 60 66 0 1 2 3 4 5 6 0 1.000 0.979 0.958 0.937 0.917 0.898 0.879 0.860 0.842 0.824 0.806 0.789 0 1.000 0.992 0.984 0.976 0.969 0.961 0.954

Days 3 0.772 0.756 0.740 0.724 0.708 0.693 0.678 0.664 0.650 0.636 0.622 0.609 1 0.946 0.939 0.931 0.924 0.916 0.909 0.902

6 0.596 0.583 0.571 0.559 0.547 0.533 0.524 0.513 0.502 0.491 0.481 0.470 Weeks 2 0.895 0.888 0.881 0.874 0.867 0.860 0.853 9 0.460 0.450 0.441 0.431 0.422 0.413 0.405 0.396 0.387 0.379 0.371 0.363 3 0.847 0.840 0.833 0.827 0.820 0.814 0.807 12 0.355 0.348 0.340 0.333 0.326 0.319 0.312 0.306 0.299 0.293 0.286 0.280 4 0.801 0.795 0.788 0.782 0.776 0.770 0.764 15 0.274 0.269 0.263 0.257 0.252 0.246 0.241 0.236 0.231 0.226 0.221 0.216 5 0.758 0.752 0.746 0.740 0.734 0.728 0.722 18 0.212 0.207 0.203 0.199 0.194 0.190 0.186 0.182 0.178 0.175 0.171 0.167 6 0.717 0.711 0.705 0.700 0.694 0.689 0.683 21 0.164 0.160 0.157 0.153 0.150 0.147 0.144 0.141 0.138 0.135 0.132 0.129 7 0.678 0.673 0.667 0.662 0.657 0.652 0.646 24 0.126 0.124 0.121 0.118 0.116 0.113 0.111 0.109 0.106 0.104 0.102 0.100 8 0.641 0.636 0.631 0.626 0.621 0.616 0.612 27 0.098 0.095 0.093 0.091 0.089 0.088 0.086 0.084 0.082 0.080 0.079 0.077 9 0.607 0.602 0.597 0.592 0.588 0.583 0.579 30 0.075 0.074 0.072 0.071 0.069 0.068 0.066 0.065 0.064 0.063 0.061 0.059 10 0.574 0.569 0.565 0.560 0.556 0.552 0.547 33 0.058 0.057 0.056 0.054 0.053 0.052 0.051 0.050 0.049 0.048 0.047 0.046 11 0.543 0.539 0.534 0.530 0.526 0.522 0.518 36 0.045 0.044 0.043 0.042 0.041 0.040 0.039 0.039 0.038 0.037 0.036 0.035 12 0.514 0.510 0.506 0.502 0.498 0.494 0.490

33P Half-Life: 25.4 days Days 012345678 9 0 1.000 0.973 0.947 0.921 0.897 0.872 0.849 0.826 0.804 0.782

Days 10 0.761 0.741 0.721 0.701 0.683 0.664 0.646 0.629 0.612 0.595 20 0.579 0.564 0.549 0.534 0.520 0.506 0.492 0.479 0.466 0.453 30 0.441 0.429 0.418 0.406 0.395 0.385 0.374 0.364 0.355 0.345 40 0.336 0.327 0.318 0.309 0.301 0.293 0.285 0.277 0.270 0.263 50 0.256 0.249 0.242 0.236 0.229 0.223 0.217 0.211 0.205 0.200 60 0.195 0.189 0.184 0.179 0.174 0.170 0.165 0.161 0.156 0.152 70 0.148 0.144 0.140 0.136 0.133 0.129 0.126 0.122 0.119 0.116 80 0.113 0.110 0.107 0.104 0.101 0.098 0.096 0.093 0.091 0.088 90 0.086 0.084 0.081 0.079 0.077 0.075 0.073 0.071 0.069 0.067 100 0.065 0.064 0.062 0.060 0.059 0.057 0.055 0.054 0.053 0.051 110 0.050 0.048 0.047 0.046 0.045 0.043 0.042 0.041 0.040 0.039 120 0.038 0.037 0.036 0.035 0.034 0.033 0.032 0.031 0.030 0.030

Figure A.1E.1 Correlation of loss of radioactivity with elapsing half-lives of an isotope. Standard Measurements, Data, and Abbreviations A.1E.3

Current Protocols in Molecular Biology Supplement 79 COUNTING EFFICIENCY The output for any radioactivity counter is given in counts (or counts per minute, cpm), which are less than the actual disintegrations from the radioisotope. Thus, it is impor- tant to take into account the counting efficiency, which is the fraction of radioactive disintegrations detected by the counter. Efficiency is determined by counting a standard sample under conditions identical to those used in the experiment. The efficiency for 125I is typically >90%, and is dependent on the geometry of the instrument. Because the detector doesn’t entirely surround the tube, a few γ rays (photons) will miss the detector. The efficiency for 32Pand35S is typically ∼80%. With 3H, the efficiency is much lower, usually 40% to 50%. This is mostly a consequence of the physics of 3H decay, which can release energy as electrons (only some of which have sufficient energy to be detected) or as neutrinos (which are not detected). The efficiency can- not be improved by better instrumentation or better scintillation fluid. In addition, the counting efficiency for 3H is reduced by the presence of any color in the counting tubes, by nonhomogeneous mixing of water and scintillation fluid, or by radioactivity that is trapped in tissue (because emitted electrons do not travel into the scintillation fluid).

SPECIFIC RADIOACTIVITY The packaging label on radioactive compounds usually states the specific radioactivity as curies per millimole (Ci/mmol). Because measurements are expressed in cpm, it is often more convenient to express the specific radioactivity as cpm/fmol. Ci/mmol can be converted to cpm/fmol using the conversion factors in Table A.1E.4. For example, if counting efficiency is 85%, a specific activity of 2500 Ci/mmol is equivalent to 4718 cpm/fmol. In many countries, specific radioactivity is provided in GBq/mmol, which can also be converted to cpm/fmol.

COUNTING ERROR Because decay is random, it is subject to sampling error. The cpm measured in a sample represents an average, with more counts in some minutes and fewer in others, and a distribution of counts that follows a Poisson distribution. There is no way to know the “real” number of counts, but a range of counts can be calculated that is 95% certain to contain the true average value. This range is known as the 95% confidence interval. The Poisson distribution explains why it is helpful to count samples longer when the number of counts is small. As an example, Table A.1E.5 shows that the confidence interval is narrower when longer counting times are used.

LITERATURE CITED Dawson, R.M.C., Elliot, D.D., Elliot, W.H., and Jones, K.M. (eds.) 1986. Data for Biochemical Research. Alden Press, London. Lederer, C.M., Hollander, J.M., and Perlman, I. (eds.) 1967. Table of Radioisotopes, 6th ed. John Wiley & Sons, New York. Shleien, B. (ed.) 1987. Radiation Safety Manual for Users of Radioisotopes in Research and Academic Institutions. Nucleon Lectern Associates, Olney, Maryland.

Radioactivity A.1E.4

Supplement 79 Current Protocols in Molecular Biology Table A.1E.4 Conversion Factors for Radioactivity Measurement of Radioactivity The SI unit for measurement of radioactivity is the becquerel: 1 Bq = 1 disintegration per second The more commonly encountered unit is the curie (Ci): 1Ci=3.7× 1010 Bq =2.22× 1012 disintegrations per minute (dpm) 1 millicurie (mCi) = 3.7 × 107 Bq = 2.22 × 109 dpm 1 microcurie (µCi) = 3.7 × 104 Bq = 2.22 × 106 dpm Conversion factors: 1 day = 1.44 × 103 min = 8.64 × 104 sec 1 year = 5.26 × 105 min = 3.16 × 107sec counts per minute (cpm) = dpm × (counting efficiency) Measurement of Dose The SI unit for energy absorbed from radiation is the gray (Gy): 1 Gy = 1 joule/kg Older units of absorbed energy are the rad (r) and roentgen (R): 1 r = 100 ergs/g = 10−2 Gy 1R=0.877rinair=0.93− 0.98 r in water and tissue The SI unit for radiation dosage is the sievert (Sv), which takes into account the empirically determined relative biological effectiveness (RBE) of a given form of radiation: dosage [Sv] = RBE × dosage [Gy] (biological effect of a dose of standard radiation[Gy]) RBE = (biological effect of a dose of other radiation[Gy]) RBE = 1 for commonly encountered radionuclides The older unit for dosage is the rem (roentgen-equivalent-man): 1rem=0.01Sv

Table A.1E.5 Example of Confidence Intervals Using Different Counting Times 1 min 10 min 100 min

Cpm 100 100 100 Total counts 100 1000 10000 95% CI of counts 81.4–121.6 938–1062 9804–10196 95% CI of cpm 81.4–121.6 93.8–106.2 98.0–102.0

Standard Measurements, Data, and Abbreviations A.1E.5

Current Protocols in Molecular Biology Supplement 79 Safe Use of Radioisotopes APPENDIX 1F

Jill Meisenhelder1 and Steve Bursik1 1The Salk Institute, La Jolla, California

ABSTRACT The pursuit of scientific knowledge has been considerably advanced by the use of biochemical molecules that incorporate radioisotopes at specific sites. The fate of these labeled molecules, and/or the radiolabeled products that result from biochemical reactions in which the parent molecule was involved, can be traced using a variety of instruments that detect radioactivity. This appendix begins with a discussion of the principles of radioactivity in order to provide the reader/user with knowledge on which to base a common sense approach to the safe use of isotopes. The characteristics of isotopes most commonly used in a molecular biology laboratory are then detailed, as well as the safety precautions and monitoring methods peculiar to each one. Detection and imaging methods used in experimental analysis are reviewed. Finally, an outline of an orderly response to a spill of radioactive material is presented. Curr. Protoc. Mol. Biol. 79:A.1F.1-A.1F.18. C 2007 by John Wiley & Sons, Inc. Keywords: radiation safety r radioactivity r isotopes r decay r shielding r monitoring r exposure r dosimeter

INTRODUCTION compliance is not optional: an institution’s The use of radioisotopes to label specific license to use radioactivity normally depends molecules in a defined way has greatly fur- on strict adherence to such rules. thered the discovery and dissection of bio- chemical pathways. The development of meth- BACKGROUND INFORMATION ods to inexpensively synthesize such tagged biological compounds on an industrial scale The Radioactive Decay Process has enabled them to be used routinely in labo- As anyone who has taken a basic chemistry ratory protocols, including many detailed in course will remember, each element is char- Current Protocols. Although most of these acterized by its atomic number (Z), defined as procedures involve the use of only microcurie the number of protons in the nucleus of that (µCi) amounts of radioactivity, some (partic- atom. Z is therefore unique to each element ularly those describing the metabolic labeling and determines the identification and chem- of proteins or nucleic acids within cells) can istry of that particular element. Isotopes of a require amounts on the order of tens of milli- given element exist because some atoms of curies (mCi). In all cases where radioisotopes each element, while by definition having the are used, depending on the quantity and na- same number of protons, have a different num- ture of the isotope, certain precautions must ber of neutrons and therefore a different atomic be taken to ensure the safety of everyone in weight or atomic mass number (A), which in- the laboratory. This unit outlines a few such dicates the total number of nucleons (protons considerations relevant to the isotopes most + neutrons). It should be noted that generally, frequently used in biological research. the number of electrons outside the nucleus In designing safe protocols for the use of ra- remains the same for all isotopes of a given dioactivity, the importance of common sense, element, so all isotopes of a given element based on an understanding of the general prin- are equivalent with respect to their chemical ciples of radioactive decay, and the importance reactivity. of continuous monitoring with a hand-held ra- Radioactive decay is defined as the spon- diation monitor (e.g., Geiger counter), cannot taneous change in the structure of an atom be overemphasized. In addition, it is critical to accompanied by the emission of energy. This take into account the relevant and applicable often results in the change of an atom of one rules, regulations, and limits of exposure. Al- element into the atom of a totally different ele- Standard though different countries have different rules, ment, a process termed nuclear transmutation, Measurements, Data, and Abbreviations

Current Protocols in Molecular Biology A.1F.1-A.1F.18, July 2007 A.1F.1 Published online July 2007 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/0471142727.mba01fs79 Supplement 79 Copyright C 2007 John Wiley & Sons, Inc. as the number of protons (or neutrons) in the stable and decay further, possibly releasing atom changes after decay. The energy released β particles. γ radiation is emitted with a can be particulate (i.e., α and β particles) or discrete energy. non-particulate (i.e., γ- and X-rays). These are Isotopic decay may involve a chain or se- the primary types of radiation encountered in quence of events rather than just a single decay. biological research. Emitted radiation is usu- Subsequent daughter products may also be ra- ally measured in units of keV (kilo-electron dioactive (unstable) and thus pose a hazard to volts) or MeV (mega-electron volts). workers. An α particle is essentially the nucleus of Following their emission, α and β radiation a helium atom, or two protons plus two neu- travel varying distances at varying speeds, de- trons. They are relatively large, heavy parti- pending on their initial energy and the atomic cles that move relatively slowly (compared to number of the material through which they are a β particle having the same amount of en- moving. The distance they actually travel be- ergy). Containing two protons, the α parti- fore interacting with the electrons or nuclei of cle has a positive charge of 2+.Witharel- another atom is termed their range and is a atively high electronic charge, it only travels defined value for each kind of material. This short distances before it readily interacts with range is usually expressed as a maximum for some other atom via coulombic forces. Emit- each type of particle, the particle’s energy, and ted α particles have discrete energies and are material. The energy and type of particulate emitted from isotopes having high mass nuclei radiation released (and therefore its potential (atomic number Z > 82; e.g., thorium or ura- range) dictates what type of shielding, if any, nium); such isotopes are not commonly used is necessary for protection against the radia- in biological research except for specific ap- tion generated by the decay of the isotope. As plications in electron microscopy and X-ray discussed previously, α radiation, even with a diffraction studies. high amount of energy, will not penetrate very In contrast to α particles, β particles are far into common materials. In fact, a sheet light, high-speed, singly charged particles. of paper can block most α radiation. There- Negatively charged β particles are essentially fore, α radiation is not considered an external electrons of nuclear origin emitted when an in- hazard, the major concern being internal ex- tranuclear neutron changes to a proton with the posure. Theoretically, γ and X-ray radiation attendant release of a neutrino (which is very can travel forever and do not have a specific weakly interacting). Release of a β particle range. Practically, however, there is a measur- thus changes the atomic number and elemen- able decrease in the intensity of γ radiation as tal status of the isotope. β radiation is emitted it penetrates greater thicknesses of material. across a spectrum of different energies, the This decrease is usually expressed as the half- most energetic being stated as Emax. The aver- value layer (HVL) or tenth-value layer (TVL) age energy can be approximated as Eave = 1/3 of a specific material (e.g., lead) and is used to 32 Emax. An example is P, with an Emax of 1.71 calculate the needed thickness of shielding. MeV and an Eave of 0.57 MeV. The neutrino When high-energy β particles released dur- is emitted with an energy equal to the differ- ing the decay of 32P encounter the nuclei of ence between the actual emitted β energy and atoms with a high atomic number, a coulombic Emax.Mostoftheβ emitters used in the biol- interaction occurs. The β particle decelerates ogy laboratory are “pure β emitters,” meaning and loses energy in the form of X-rays. Such there is no other radiation emitted besides the X-rays are termed bremsstrahlung radiation β particle and the neutrino. (German for “breaking radiation”); they are γ radiation exhibits both particle and wave detectable using most radiation survey meters, properties, and its wavelength falls within the especially those designed for the detection of range of X-ray wavelengths. Physically, there γ or X-rays. The amount of bremsstrahlung is no difference between γ and X-ray radia- radiation produced is directly proportional to tion. The only difference is their physical ori- both the energy of the incident β particle and gin. γ radiation is defined as that originating the atomic number, Z, of the absorber. Hence, from an atomic nucleus, while X-ray radiation much more bremsstrahlung is produced by 32P 35 35 originates from the electron cloud surround- β radiation than S β radiation (Emax for Sis ing the nucleus. Unlike β particle emission, 0.167 MeV) when incident on the same mate- the emission of γ radiation by itself produces rial, and more is produced by 32P β radiation an isotopic change rather than an elemental when incident on a high-Z material such as Safe Use of one; however, the resultant nuclei may be un- lead compared to plastic. Radioisotopes A.1F.2

Supplement 79 Current Protocols in Molecular Biology α, β, and γ emissions all have the poten- obtained by multiplying the number of rads by tial, upon encountering an atom, to interact a “quality factor,” which is based on the type with and ionize the atom. Thus, these three of ionizing radiation delivering the dose. For types of emissions are called ionizing radia- β particles and γ or X-rays this factor is 1 and tion. The formation of such ions may result therefore rems and rads are equal for β radia- in the perturbation of biochemical processes: tion. In contrast, the quality factor associated therein lies the health concern associated with with α particles is 20, so a dose of 1 rad due radioactivity! to α particles would be recorded as 20 rem. Thus, dose equivalent is based on both physi- Units Used for Radioactivity cal and biological factors. The dose equivalent There are several measurable properties is the most meaningful quantity used for radi- used to describe the amount and physical ef- ation protection purposes and is the unit used fects of ionizing radiation: activity, exposure, to record dosimeter badge readings. There are dose and dose equivalent. 100 rems in a sievert, the unit of dose equiva- Activity is the amount of radioactive mate- lent used in most countries other than the U.S. rial in a sample and it is measured in units of curies (Ci) or becquerels (Bq). A curie by def- inition is that amount of radioactive material Measuring Exposure to Ionizing that will produce 3.7 × 1010 disintegrations Radiation (ions) per second. This was originally deter- The radiation dose received by materials mined as the number of disintegrations that (cells, scientists, etc.) near a radioactive source occur during the radioactive decay of 1 g of depends not only on the specific type and en- radium-226. The becquerel is defined as 1 dis- ergy of the radiation absorbed, but also on the integration per second. In biological research, subject’s distance from the source, the exis- the curie is a very large unit and the becquerel tence of any intervening layers of attenuat- a very small unit, so prefixes are added to ing material (shielding, clothing, etc.), and the these roots to express the activity amounts be- length of time spent in the vicinity of the radi- tween these two extremes that are commonly ation source. To best measure doses to person- used, i.e., microcurie (µCi), Megabecquerel nel, everyone working with or in close prox- (MBq). Several useful tables on radioactivity imity to radioactive sources should wear the and physical characteristics of radioisotopes appropriate type of radiation dosimeter badge can be found in APPENDIX 1E. (in addition to using a portable radiation mon- Exposure is defined as that amount of ion- itor that can give an immediate indication of ization (measured as electrical charge) pro- the presence of radiation). This is normally duced in a particular volume of air at standard a requirement (not an option) for compliance temperature and pressure (STP) by the passage with an institution’s radioactive materials li- of γ or X-ray radiation. This unit is only de- cense. Such badges are usually furnished by fined for γ or X-ray radiation. The exposure the radiation safety department, collected at unit is the X unit, which is slowly supplant- regular intervals, and sent for processing to ing the roentgen (R) for expressing this prop- a contracted company. Most institutions use erty. One X unit is 1 coulomb per kilogram either TLDs (thermoluminescent dosimeters) of air. This property can be measured directly or OSLDs (optically stimulated luminescent with ionization chamber-based radiation sur- dosimeters). Both devices use crystals of cal- vey meters. cium fluoride, lithium fluoride, or aluminum Dose is the property that describes the de- oxide; the electron structure of these crys- position of energy. It is defined as the amount talline materials is altered following exposure of deposited radiation energy per mass unit of to ionizing radiation. During processing, stim- absorber. A deposition of 100 ergs in 1 gram of ulation by either heat (for TLDs) or laser light material is equivalent to 1 rad. There are 100 (for OSLDs) will cause the crystals to lumi- rads in 1 gray, another common unit of dose. nesce, and the intensity of luminescence is Defined doses for isotopes commonly used in directly proportional to the dose of ionizing the molecular biology laboratory are shown radiation that has been absorbed. The results in Table A.1F.1. Dose is important, but only are then compared with known controls to considers physical factors. determine the actual dose equivalent. Differ- The property of dose equivalent is most cor- ent types of badges are sensitive to different Standard rectly used to describe the potential for damage types of radiation. Usually the Safety Office Measurements, to an irradiated individual. The unit for dose will determine what badge is best to wear Data, and Abbreviations equivalent is the rem. The number of rems is while working in a particular facility. Workers A.1F.3

Current Protocols in Molecular Biology Supplement 79 Table A.1F.1 Physical Characteristics of Commonly Used Radionuclidesa

Max range Dose 10 cm Specific Decay Energy Isotope Half-life (cm) in from 1 mCi activity, 100% Typical use in laboratory mode (MeV)b air/tissue point source pure (Ci/mg)

3Hc 12 years β- 0.0186 0.42/0 0 mrad/hr 9.6 Cell proliferation assays (3H-thymidine); tagging cellular proteins (3H-amino acids) 14Cc 5730 β- 0.156 22/0.027 600 mrad/hr 0.0044 Tagging cellular proteins years (14C-amino acids) 32Pd 14 days β- 1.71 620/0.08 4070 mrad/hr 287 Probes for northern and Southern blots (α-dNTPs); in vitro kinase reactions (γ-ATP); metabolic labeling of cellular proteins (ortho-32P) 33Pc 25 days β- 0.249 49/0.06 2000 mrad/hr 156 Probes for northern and Southern blots (α-dNTPs) 35Sc 87 days β- 0.167 24/0.03 625 mrad/hr 43 Metabolic labeling of cellular proteins or in vitro translation (35S-methionine/cysteine) 125Ie 60 days γ 0.027-0.035 HVL = 0.2 15 mrem/hr 17 Labeling of cell surface mm lead proteins or of purified proteins in vitro (free 125I); immunoblot detection reagent (125I-protein A) aTable compiled based on information in Lederer et al. (1967), Shleien (1987), and the Princeton Radiation Safety Manual (http://web.princeton.edu/ sites/ehs, see radioisotope fact sheets). bA 100 W light bulb burning for 1 hr uses 2.2 × 1018 MeV. The energy of visible light is 1.8 to 3.1 eV. cShielding is not needed for activity amounts typically used in the laboratory. dRecommended shielding for 32P is clear acrylic plastic (up to 1 cm thick for mCi amounts). eRecommended shielding for 125I is lead foil or (for mCi amounts) a leaded acrylic workstation.

should be trained to always wear their dosime- to the action limit for extremities set by the ter badge on the outside of their laboratory institution when working with multiple milli- coat, chest high, facing toward their work, as curies of 32P. U.S. Federal annual dose lim- the results will read low if the badge has any its are as follows: 5,000 mrem for the whole layers of material (e.g., laboratory coats, jack- body (trunk and upper extremities); 50,000 ets, and pocket material) covering it up. Preg- mrem for the skin, extremities, and thyroid; nant women are required to be educated and and 15,000 mrem for the lens of the eye. Sci- given the option to wear a dosimeter to bet- entists believe that there would not be any bi- ter monitor the dose equivalent to their de- ological effect in an individual exposed to less veloping fetus. When working with >1 mCi than these limits during each year of their pro- of high-energy β emitters (32P) or with any fessional life (40 years used). γ/X-ray emitting isotope (125I), there is most What is known about the risks to humans likely an institutional license requirement for after exposure to low levels of radiation—i.e., researchers to wear a ring badge to measure levels that would be received when briefly dose to the unshielded (though gloved!) fin- handling small amounts (µCi or mCi) of gers and hands (extremities). The limit for radioactivity? Unfortunately, the nature of the “acceptable” exposure to the extremities is ten problem precludes the ability of scientists times more than the limit for the whole body. to perform controlled studies to make this Safe Use of However, the dose equivalent recorded with determination, and a complete consensus has Radioisotopes dosimeter rings may be significant compared not been reached. However, most experts A.1F.4

Supplement 79 Current Protocols in Molecular Biology think the use of a linear model extrapolated arate from the rest of the laboratory. Many in- down from the quantitatively determined stitutions require that such work be performed effects of high doses of ionizing radiation is in a designated “hot lab”; however, if many a conservative means to determine the risk people in the laboratory routinely use radioiso- per rem. These studies make use of data topes, it is less than feasible to move them all obtained from studies of personnel exposures into what is usually a smaller space. No mat- received during radiation accidents, medical ter where an individual is working, it is his or treatments, occupational exposures, and—the her responsibility to monitor the work area and largest study group—survivors of the atomic ensure his or her own safety and the safety of bombs dropped on Hiroshima and Nagasaki those working nearby. To protect bystanders, during WWII. Genetic risks to subsequent remember that the intensity of radiation from generations are estimated using data from a small source (moving through air) falls off animal experiments and the families of atomic in proportion to the square of the distance. bomb survivors. However, since each form of Thus, if standing 1 foot away from a source for extrapolation is subject to caveats, and given 5 min would result in an exposure of 45 mrem, that predictions based on such extrapolations standing 3 feet away for the same amount of cannot be perfect, most health and safety time would result in an exposure (1/3)2 of 45 personnel aim for radiation exposure levels to mrem, or about 5 mrem. This factor is also be ALARA or “as low as reasonably achiev- relevant when considering the storage of large able.” An extensive discussion of both the amounts of radioactivity, particularly 125Ior studies and statistics on which federal annual 32P, as sometimes radiation cannot be com- dose limits are based, which is updated on a pletely attenuated. regular basis, may be found in the Biological Effects of Ionizing Radiation series (BRER, Shielding: The great wall 2006; available online in open book form at When handling radioactive samples, it may http://www.nap.edu/books/030909156X/html). be necessary to work behind shielding. When shielding is properly used, it will successfully MINIMIZING EXPOSURE minimize researcher (and neighbor) exposure Minimizing exposure to ionizing radia- to radioactive materials. However, when used tion can be accomplished by adjusting several improperly it can lead to worker fatigue, awk- parameters of the exposure: minimizing the ward movements, and a higher chance of number and duration of exposures, increas- spillage. Set up your work station in advance to ing the distance between the researcher and make sure you are comfortable with the physi- the source, and using appropriate shielding be- cal layout of your equipment and shielding. If tween the researcher and the source. feasible, start with small amounts of activity. With valuable experience and increased com- Time is of the essence fort working with these small activity amounts, When designing any experiment using ra- the transition to using shielding for higher ac- dioactivity, prudent efforts should be made to tivity amounts will be easier to make. When limit the time spent directly handling vials or using mCi amounts of 32P, shielding will al- tubes containing radioactive material or work- ways be needed to minimize worker exposure. ing in close proximity to radioactive materials. As mentioned before, the energy of the par- This includes minimizing the chance of error ticle(s) released during the decay of an isotope so as not to have to unnecessarily repeat an determines what type of shielding, if any, is experiment. Work at a pace that is comfort- appropriate. β particles released during the de- able for you—not dawdling, but also not so cay of 14C and 35S possess roughly ten times fast as to cause spillage or error! Have every- the energy of those released when 3H decays. thing needed for the experiment ready at hand However, all three of these isotopes emit β before the radioactivity is introduced into the particles of relatively low energy, which do work area. This includes materials, equipment, not travel very far in air and cannot penetrate and a thorough knowledge of the procedure through solid surfaces. Therefore, these iso- being performed. The more time spent famil- topes are not considered an external hazard iarizing yourself with your protocol, the more and shielding barriers are not necessary when smoothly the work will go. working with them. The major health hazard from these isotopes is internal exposure, which Standard Keep your distance can occur through accidental ingestion, inhala- Measurements, When possible, experiments involving ra- tion, absorption through the skin, or introduc- Data, and Abbreviations dioactivity should be performed in an area sep- tion through the skin by a wound. A.1F.5

Current Protocols in Molecular Biology Supplement 79 β particles released during the decay of value layer (HVL) measurement for each type 32P may have a 10-fold higher energy than of shielding material and γ/X-ray energy. The those released from 14C and may pose a HVL for 125I is 0.02 mm of lead. This small very real concern to workers, especially if thickness of lead is sufficient to shield most multi-mCi amounts of activity are being han- activity amounts of 125I used in the laboratory. dled. (One potential biological effect is induc- When using prelabeled kits, shielding is not tion of cataracts in the unshielded eye; how- needed, as the activity used in these kits is ever, the threshold dose for cataracts is on typically very small (<10 µCi). the order of 500 rem, which could only be delivered in a laboratory setting by using mCi amounts of 32P without adequate shielding GENERAL PRECAUTIONS over a span of many years.) As explained Before going on to a discussion of specific previously, the fact that these high-energy β precautions to be taken with individual iso- particles can potentially generate significant topes, a short list of general precautions to be amounts of bremsstrahlung radiation is the rea- taken with all isotopes seems pertinent. son that low-Z (atomic number) materials are 1. Know the rules. Be sure that each in- used as the primary layer of shielding for 32P β dividual is authorized to perform an approved radiation. Water, glass, and plastic are suitable procedure using a particular isotope and activ- low-Z materials (as opposed to lead). Obvi- ity amount in the approved work area. ously, water is unsuitable as a shielding layer 2. Don the appropriate apparel. When- for work on the bench, although it does a rea- ever working at the laboratory bench, it is sonable job when samples are incubating in a good safety practice to wear a laboratory coat water bath. Shields made from a thickness of for protection; when using radioactivity, wear- glass sufficient to stop these particles would ing a laboratory coat is imperative. Dispos- be extremely heavy and cumbersome (as well able paper/synthetic coats of various styles as dangerous if dropped). Fortunately, plastic are commercially available: at $4 each these or acrylic materials, variously called Plexiglas, may be conveniently thrown out if contami- Perspex, or Lucite, are available for shielding nated with radioactivity during an experiment, against 32P β radiation. Shields, sample stor- rather than held for decay as might be prefer- age boxes, waste container boxes, and sam- able with cloth coats costing ∼$30 each. As ple racks constructed of various thicknesses of an alternative, disposable sleeves can be pur- Plexiglas are necessary equipment in labora- chased and worn over the usual cloth coat. tories where 32P is used. For plastic or acrylic Other necessary accessories include radiation material, a thickness of about 0.64 to 1.0 cm is dosimeter badges, protective eyewear, shoes adequate for shielding up to 5 mCi of 32P. To that cover the top of the foot, and two pairs of shield against bremsstrahlung radiation when gloves. When one of the outer gloves becomes using higher activity amounts of 32P, it is nec- contaminated, it is easy to slowly peel it off, essary to add a layer of high-Z material (such replace if necessary with another glove, and as 0.38 to 2.29 mm lead) to the outside of the continue working with only minor interrup- Plexiglas shield that is opposite the radioactive tion. Removal of contaminated gloves should source (Klein et al., 1990). always be performed over a bench or waste γ/X-rays released during the decay of 125I container so that microdroplets of contamina- will easily penetrate the plastic materials used tion do not fall on the floor and get tracked to shield β particles from 32P; this radiation about! must be reduced in intensity using a high-Z 3. Protect the work area as well as the material, such as lead. Lead foil of varying workers. Laboratory bench tops and the bases thicknesses (0.76 to 1.0 mm) can be purchased of any shields should be covered with a dis- in rolls and can be cut and molded to cover any posable, preferably absorbent, layered paper container, or taped to a Plexiglas shield (used sheet. Blue absorbent pads (“hospital diapers”) in this instance for support). The latter arrange- work quite well. ment has the obvious disadvantage that it is im- 4. Use appropriately designated equip- possible to see what one is doing through the ment. It is very convenient, where use justifies shield. For routine shielding of manipulations the expense, to have a few adjustable pipettors involving mCi activities of 125I, it is useful dedicated and labeled for use with each par- to purchase a lead-impregnated, transparent, ticular isotope. Likewise, it is good practice Plexiglas shield (which can be very heavy as to use only certain labeled centrifuges and mi- Safe Use of well as relatively expensive). When deciding crocentrifuge rotors for radioactive samples so Radioisotopes how thick is “thick enough,” consult the half- that all of the rotors in the laboratory do not A.1F.6

Supplement 79 Current Protocols in Molecular Biology become contaminated. Although such equip- before you begin, during, and after all pro- ment should be cleaned after each use, com- cedures. The more frequently fingers, hands, plete decontamination is often not possible. A relevant equipment, and your work area are few pipettors or a single microcentrifuge can monitored, the more quickly a spill or glove easily be stored (and used) behind appropriate contamination will be detected. Timely detec- shielding. Contamination of the insides and tion will keep both the potential spill area and tip ends of pipettors can be greatly reduced by the cleanup time to a minimum. While it is using tips supplied with internal aerosol bar- tempting to cover the monitor’s detector tube riers such as those used for PCR reactions. with Parafilm to protect it from contamination, To prevent contamination of the outside of the remember that this will prevent the detection pipettor barrel, simply wrap the hand-grip in of low-energy β radiation from 35S and 14C! Parafilm, which can be discarded later. Because the low-energy β emitter 3H cannot 5. Know where to dispose of radioactive be detected at all using these monitors, obtain- waste, both liquid and solid. Most institu- ing wipe samples of the bench and equipment tions require that radioactive waste be seg- and subsequently counting them with a liquid regated by isotope and physical form. This scintillation counter is necessary to ensure that is done not only so that appropriate shield- contamination of the work area did not occur. ing can be placed around waste containers, 8. Clean up contamination as soon as but so that some waste can be allowed to de- possible after discovery! If contamination is cay prior to disposal through normal (nonra- discovered, it is to everyone’s benefit (yours dioactive) trash methods. With a decreasing and your neighbors’) that it be cleaned up as number of radioactive waste disposal facilities soon as possible. This will prevent the inad- able or willing to accept radioactive waste for vertent spread of contamination to other areas burial (and a concomitant increase in dumping (and people) in the laboratory. To wait over a charges from those that still do), the practice weekend until Monday, or even one evening, of on-site decay can save an institution thou- to clean up contamination can invite disaster. sands of dollars a year in disposal charges. A relatively small spill can turn into a large With this in mind, the volume of radioactive mess if tracked about. All things considered, waste present at your institution can be min- it is imperative to take the time to clean up a imized by surveying all items before placing spill immediately. them into the radioactive waste. This will re- sult in lower costs associated with the disposal SPECIFIC PRECAUTIONS of radioactive materials. The following sections describe precau- 6. Label your label! It is only common tions to be taken when working with individ- courtesy (as well as common sense) to alert ual isotopes in specific forms. Although the coworkers to the existence of anything and sections dealing with 35S- or 32P-labeling of everything radioactive that is left where they proteins in intact cells are presented in terms may come in contact with it! A simple piece of of mammalian cells, most of the instructions tape affixed to the sample box with the inves- are also pertinent (with minimal and obvious tigator’s name, the activity amount and type modifications) to the labeling of proteins in of isotope, and the date written on it should other cells (bacterial, insect, plant, etc.). suffice. Yellow hazard tape printed with the international symbol for radioactivity is com- mercially available in a variety of widths for Working with 3H this purpose. Tritiated compounds used in the molecular 7. Monitor for radioactive contamination biology laboratory include 3H-labeled thymi- early and often. It is imperative that each lab- dine (used in cell proliferation assays) and oratory authorized to use radioactive material 3H-labeled amino acids (used to label newly have access to the use of a portable radiation synthesized proteins). As discussed above, the survey meter. Some meters are more suited for β radiation resultant from the decay of tritium β detection and some are more suited for γ is of such weak energy that no type of shield- detection. Keep the appropriate survey meter ing is necessary to protect the scientist during nearby; switch it on before touching anything the experiment. In fact, these β particles can- on your laboratory bench to avoid contaminat- not even be detected using a typical hand-held ing the switch on the meter. Always check the Geiger-Muller¨ monitor (also known as Geiger Standard batteries before using a survey meter! Use a counter or GM monitor). Therefore, to avoid Measurements, monitor with an adequate detector efficiency accidental ingestion or absorption through un- Data, and Abbreviations (β detector for 35S and 32P; γ detector for 125I) intentional/unrealized contact, it is imperative A.1F.7

Current Protocols in Molecular Biology Supplement 79 that the researcher perform wipe tests of the ex- controlled area such as a mini-hood equipped perimental area and equipment used to deter- with a charcoal filter. This charcoal filter will mine if any contamination exists. These wipe become quite contaminated and should be tests most often consist of both random and changed every few months. If such an area is specific (most-likely candidate) swipes of sur- not available, the stock vial should be thawed faces with a paper filter that is subsequently using a needle attached to a charcoal-packed counted, with fluor, in a liquid scintillation syringe to vent and trap the volatile compound. counter to determine if any 3H is/was present. Anyone who has ever added 35S-labeled amino acids to dishes of cells for even short Working with 14Cand35S periods knows that the incubator(s) used for These two isotopes are used to label amino such labeling may quickly become highly con- acids and thus proteins; 14Cisalsousedtola- taminated with 35S. Such contamination is not bel various reagents used in assays as well as limited to the dish itself, nor to the shelf on molecular weight standards to run on protein which the dish was placed. Rather, the radioac- gels. As discussed above, the β radiation gen- tive component’s solubility in water allows it erated during 14Cor35S decay is not strong to circulate throughout the moist atmosphere enough to make additional shielding neces- of the incubator and contaminate all of the in- sary. The risk associated with both these iso- side surfaces of the incubator. For this reason, topes comes primarily through their ingestion in laboratories where such metabolic labeling and subsequent concentration in various target is routine, it is highly convenient to designate organs, depending on the compound to which one incubator to be used solely for working the radioisotope is attached. Although will- with 35S-labeled samples. Such an incubator ful ingestion of either isotope seems unlikely, can be fitted with a large honeycomb-style fil- accidental or unknowing ingestion may occur. ter the size of the incubator shelf, made of Additionally, the half-life of 14C is 5730 years, pressed, activated charcoal. These filters are so do not spill this! available from local air-quality-control com- panies. Such a filter will quickly become con- Using 35S to label cellular proteins and taminated with radioactivity and should there- proteins translated in vitro fore be monitored and changed as necessary As reported several years ago (Meisen- (e.g., every three months if the incubator is helder and Hunter, 1988), 35S-labeled methio- used several times a week). The water used nine and cysteine, which are routinely used to to humidify the incubator will also become label proteins synthesized in intact cells and quite “hot” (contaminated with radioactivity); by in vitro translation, break down chemically keeping the water in a shallow glass pan on to generate a volatile radioactive component. the bottom of the incubator makes it easy to Because this breakdown occurs independent change after every use, thus preventing the of cellular metabolism, the radioactive com- contamination from accumulating. Even with ponent is generated to the same extent in the charcoal filter and water as absorbents, the stock vials as in cell culture dishes. The shelves, fan, and inner glass door of the incuba- process seems to be promoted by freezing tor will become contaminated, as will the tray and thawing 35S-labeled materials. The ex- on which the cells are carried and incubated. act identity of this component is not known, Routine wipe tests and cleaning when neces- although it is probably SO2 or CH3SH. sary will help to minimize potential spread of What is known is that it dissolves readily in this contamination. water and is absorbed by activated charcoal or If such work is done infrequently or there copper. is not a “spare” incubator, dishes of cells can The amount of the volatile radioactive com- be placed in a box during incubation. This box ponent released, despite stabilizers added by should be made of plastic, which is generally the manufacturers, is about 1/8000 of the to- more easily decontaminated than metal. Along tal radioactivity present. The amount of this with the dishes of cells, a small sachet made radioactivity that a scientist is likely to inhale of activated charcoal wrapped loosely in tis- while using these compounds is presumably sue (Kimwipes work well) should be placed in even smaller. Nevertheless, such a component the box. If the box is sealed, it will obviously can potentially contaminate a wide area be- need to be gassed with the correct mixture of cause of its volatility, and would tend to con- CO2; otherwise, small holes can be incorpo- centrate in target organs. Thus, it is advisable rated into the box design to allow equilibra- Safe Use of to thaw vials of 35S-labeled amino acids in a tion with the incubator’s atmosphere. In either Radioisotopes A.1F.8

Supplement 79 Current Protocols in Molecular Biology Figure A.1F.1 Plexiglas shielding for 32P. ( A) Two portable shields (L and T design) made of 0.5-in. (12.5-mm) Plexiglas. Either can be used to directly shield the scientist from the radioactivity being used. Turned on its side, the L-shaped shield can be used to construct two sides of a cage around a temporary work area, providing shielding for workers directly across from or to the sides of the person working with 32P. ( B) Tube rack for samples in microcentrifuge tubes. (C) Tube holder for liquid waste collection. case, the incubator used for the labeling should basal cells resulting from a skin contamina- be carefully monitored for radioactivity after tion of 1 µCi/cm2 is 9200 mrads/hr (Shleien, each experiment. 1987). Such a skin contamination could eas- ily be obtained through careless pipetting and Working with 32P the resultant creation of an aerosol of radioac- tive microdroplets, because the concentration µCi amounts of 32P of a typical stock solution of labeled nucleotide The amount of 32P-labeled nucleotide used maybe10µCi/µl. to label nucleic acid probes for northern or For proper protection during these types of Southern blotting is typically under 250 µCi, experiments, besides the usual personal attire and the amount of [γ−32P]ATPusedfor (glasses, gloves, coat, closed-toe shoes, and in vitro phosphorylation of proteins does not ring and lapel dosimeter badges) it is nec- usually exceed 50 µCi for a single kinase reac- essary to use some form of Plexiglas shield tion (or several hundred µCi per experiment). (Fig. A.1F.1A) between the body and the However, given the time spent on such experi- samples. Check the level of radiation com- ments, handling even these small amounts can ing through the outside of the shield with result in a measurable dose equivalent if proper a portable monitor to ensure that the thick- Standard shielding is not used. With no intervening ness of the Plexiglas is adequate. Hands can Measurements, shielding, the dose rate 1 cm away from 1 mCi be shielded from some exposure by placing Data, and Abbreviations 32P is 200,000 mrads/hr; the local dose rate to the sample tubes in a solid Plexiglas rack A.1F.9

Current Protocols in Molecular Biology Supplement 79 (Fig. A.1F.1B), which is also useful for trans- When this figure is multiplied by the number porting samples from the bench to a centrifuge of dishes necessary per sample, and the num- or water bath. ber of different samples in each experiment, These experiments often include an incuba- the amount of 32P used in one experiment tion step performed at a specific temperature, can easily reach 25 mCi or more. Because so usually in a water bath. Although the water sur- much radioactivity is used in the initial label- rounding the tubes or hybridization bags will ing phase of such experiments, it is necessary effectively stop β radiation, shielding should for a researcher to take extra precautions in be added over the top of the tubes where there order to adequately shield him or herself and is no water (e.g., using a simple flat piece of coworkers. Plexiglas). If the frequency of usage justifies When adding 32P label to dishes of cells, it the expense, an entire lid for the water bath is important to work as rapidly and as smoothly can be constructed from Plexiglas. When hy- as possible. An important contribution to the bridization reactions are performed in bags, speed of these manipulations is to have ev- care should be taken to monitor (and shield) erything that will be needed at hand before the apparatus used to heat-seal the bags. It is even introducing the label into the work area. obviously also important to ensure that the wa- Prepare the work area in advance, arranging ter in the bath does not become contaminated shielding and covering the bench with blue di- by leakage from hybridization samples. apers. Set out all necessary items, including The waste generated during the experi- pipettors and tips needed, a portable detec- ments should also be shielded. It is convenient tion monitor, extra gloves, and a cell house to have a temporary, satellite waste container (Fig. A.1F.2A). right on the bench. Pipet tips and other solid Research using this much radioactivity waste can be discarded into a Plexiglas box should be done behind a Plexiglas shield at lined with a plastic bag and placed behind least 3/4 in. (2 cm) thick; the addition of a the shield. This bag can then be emptied into layer of lead to the outside lower section of the appropriate shielded laboratory waste con- this shield to stop bremsstrahlung radiation tainer when the experiment is done. Liquid is also needed. If one shield can be dedi- waste can be pipetted into a disposable tube cated to this purpose at a specific location, set in a stable rack or holder behind the shield a sheet of lead 4 to 6 mm thick can be perma- (Fig. A.1F.1C). nently screwed to the Plexiglas (Fig. A.1F.2B). When radiolabeled probes or proteins must However, this lead makes the shield extremely be gel-purified, it may be necessary to shield heavy and therefore less than portable. If space the gel apparatus during electrophoresis if the constraints do not permit the existence of a samples are particularly hot. Be advised that permanent labeling station, a layer or two of the electrophoresis buffer is likely to become thick lead foil can be taped temporarily to the very radioactive if the unincorporated label is outside of the Plexiglas shield. allowed to run off the bottom of the gel; check Again, each worker should take care to with radiation safety personnel for instructions shield not only him or herself, but also by- on how to dispose of such buffer. It is also pru- standers on all sides. Handling of label should dent to check the gel plates with a radiation be done away from the central laboratory, if survey meter after the electrophoresis is com- possible, to take maximum advantage of dis- pleted since they can become contaminated as tance as an additional means of dose reduction. well. It is also advisable not to perform such exper- iments in a tissue culture room or any other mCi amounts of 32P room that is designed for a purpose vital to the In order to study protein phosphorylation whole laboratory. An accident involving this in intact mammalian cells, cells in tissue cul- much 32P would seriously inconvenience fu- ture dishes are incubated in phosphate-free ture work in the area, if not make it altogether medium with 32P-labeled orthophosphate for uninhabitable! If care is taken to minimize the a period of several hours or overnight to label amount of time the dish of cells is open when the proteins. The amount of 32Pusedinsuch adding the label, use of a controlled air hood procedures can be substantial. Be sure to cal- to prevent fungal or bacterial contamination of culate and pipet the actual activity, rather than the cells should not be necessary. just partitioning the label into equal volume In the course of doing experiments to deter- aliquots. Cells are normally incubated in 1 to mine which hand receives the most exposure Safe Use of 2mCiof32P/ml labeling medium; for a 6-cm during such cell labeling procedures, extrem- Radioisotopes dish of cells, 2.5 to 5 mCi 32P may be used. ity exposure was shown to vary as much as A.1F.10

Supplement 79 Current Protocols in Molecular Biology Figure A.1F.2 (A) Box for cell incubation (a “cell house”). (B) Stationary leaded shield. (C) Sample storage rack and box made of 0.5-in. Plexiglas. (D) Box for solid waste collection made of 0.5-in. Plexiglas. ID, interior dimension.

Standard Measurements, Data, and Abbreviations A.1F.11

Current Protocols in Molecular Biology Supplement 79 Figure A.1F.3 Use of Plexiglas dish shields for 32P reduces extremity exposure.

ten-fold depending on which finger the sertion of the dishes) and have a handle on dosimeter ring was worn on, with the index top (for safe carrying) make ideal cell houses finger of the left hand receiving the most ex- (Fig. A.1F.2A). A Plexiglas door that slides posure for a right-handed person (Bursik et al., into grooves at the open end is important to 1999). As would be expected, the most expo- prevent dishes from sliding out if the box is sure is received as the worker adds label to tilted at all during transport. If this door is the dishes of cells and as the cells are lysed only two-thirds the height of the house wall, (see below). In order to mitigate this extremity the open slot thus created will allow equilibra- exposure, Plexiglas dish covers (Fig. A.1F.3) tion of the CO2 level within the house with can be used to shield each individual dish: the that in the incubator. Obviously, this slot will tissue culture dish fits snugly into the bottom also allow a substantial stream of radiation to Plexiglas piece while the top Plexiglas piece pass out of the cell house, so the house should is joined to the top of the tissue culture dish be carried and placed in the incubator with its using tape so that the two lids together can door facing away from the worker (and oth- be handled as one unit. Tissue culture dishes ers)! The use of Plexiglas dish covers adds of cells are fitted/taped into the Plexiglas dish considerable bulk to the dishes of cells, and covers immediately before adding the 32P. As larger cell houses designed with handles on the top and bottom pieces of the dish covers their sides and a hinged lid are more easily do not form a seal, the medium can equilibrate handled (Fig. A.1F.3). with the CO2 of the incubator for proper pH Following incubation with label and any adjustment. Use of such dish covers reduces treatments or other experimental manipula- extremity exposure by 8- to 10-fold, despite tions, the cells are usually lysed in some type the stream of radiation that passes through the of detergent buffer. It is during this lysis pro- small crack between the top and bottom. cedure that a worker’s hands will receive their Once the label has been added to the dishes greatest exposure to radiation, because it is of cells (and whether or not one is using the necessary to handle pipettors directly over dish covers discussed above), they will also open cell dishes for a period of several min- need to be shielded for transport to and from utes. It is therefore very important to stream- the incubator and other work areas. Plexi- line this procedure and use shielding when- Safe Use of glas boxes that are open at one end (for in- ever possible. If the cell lysates must be made Radioisotopes A.1F.12

Supplement 79 Current Protocols in Molecular Biology at 4◦C, as required by most protocols, work- tor and clean out the centrifuge after each use ing on a bench in a cold room is preferable so contamination does not accumulate. to placing the dishes on a slippery bed of ice. The amount of 32P taken up by cells during In either case, make the lysate using the same the incubation period varies considerably, de- sort of shielding (with lead, if necessary) that pending on the growth state of the culture as was used when initially adding the label. Using well as on the cell type and its sensitivity to disposable transfer pipets, remove the labeling radiation. This makes it difficult to predict the medium, and any solution used to rinse unin- percentage of the radioactivity initially added corporated radioactivity from the cells, into a to the cells that becomes incorporated into the small tube held in a solid Plexiglas holder (Fig. cell lysate; however, this figure probably does A.1F.1C). The contents of this tube can later not exceed 10%. Thus, the amount of radioac- be poured into the appropriate liquid waste re- tivity being handled decreases dramatically ceptacle. If possible, it is good practice to keep after lysis, making effective shielding much this high-specific-activity 32P liquid waste sep- simpler. Nonetheless, at least ten times more arate from the lower-activity waste generated radioactivity is still involved compared to other in other procedures so that it can be removed sorts of experiments! It is easy to determine if from the laboratory as soon as possible fol- the shielding is adequate—just use both β and lowing the experiment in a specially designed γ survey meters to measure the radiation pass- shielded box. The Radiation Safety Officer ing through the shielding. As a rule of thumb, should be asked to remove this high-activity if the meter reads more than 5000 cpm, addi- waste as soon as possible. If it is necessary tional shielding is needed. Again, be sure that to store it in the laboratory for any time, the people working nearby (including those across shielding for the waste container should also the bench) are also adequately shielded. It is include a layer of lead. sometimes necessary to construct a sort of cage The solid waste generated in the lysis part of Plexiglas shields around the ice bucket that of these experiments (pipet tips, disposable contains the lysates. pipets, cell scrapers, and dishes) is very hot and At the end of the day or the experiment, should be placed immediately into a shielded it may be necessary to store radioactive container to avoid further exposure to the samples; in some experiments, it may be hands. A hinged Plexiglas box (Fig. A.1F.1D), desirable to save the cell lysates. These very placed to the side of the shield and lined with hot samples are best stored in tubes placed in a plastic bag, will safely hold all radioac- solid Plexiglas racks that can then be put into tive waste during the experiment, and is light Plexiglas boxes (Fig. A.1F.2C). Such boxes enough to be carried easily to the main ra- may be of similar construction to the cell dioactive waste container, where the plastic houses described above, but they should have bag (and its contents) can be dumped after the a door that completely covers the opening. Be experiment is completed. If the lid of the box sure to check for γ radiation coming through protrudes an inch or so over the front wall, it these layers and add lead outside the box if can be lifted using the back of the hand, thus necessary. decreasing the possibility of spreading con- tamination with hot gloves. Working with 33P When scraping the cell lysates from the dishes, it is good practice to add them to Using 33P-labeled nucleotides to label microcentrifuge tubes that are shielded in a nucleic acid probes or proteins solid Plexiglas rack; this will help to further Several of the major companies that manu- reduce the exposure to which the hands are facture radiolabeled biological molecules also subjected. At this point, the lysates are usu- sell nucleotides labeled with 33P (both α and ally centrifuged at high speed (10,000 × g) γ structural forms). 33P offers a clear advan- to clear them of unsolubilized cell material. tage over 32P with respect to ease of han- Use screw-cap tubes for this clarification step, dling, because the maximum energy of the as these will contain the labeled lysate more emitted β radiation is between that of 35S securely than flip-top tubes, which may open and 32P and does not require as much Plex- during centrifugation. No matter what type of iglas or lead thickness as is needed for 32P. tube is used, the rotor of the centrifuge of- In fact, the β radiation emitted (Emax=0.248 ten becomes contaminated, most probably be- MeV) can barely penetrate through two pairs Standard cause tiny drops of lysate (aerosol) initially of gloves and the outer dead layer of skin, so Measurements, present on the rim of the tubes are spun off the external exposure hazard associated with Data, and Abbreviations during centrifugation. It is important to moni- even millicurie amounts of 33P is minimal (as A.1F.13

Current Protocols in Molecular Biology Supplement 79 reported in the DuPont NEN product sorbed, or inhaled iodine is concentrated in brochure). Gel bands visualized on autoradio- the thyroid, a portable γ monitor should be graphs of 33P-labeled compounds are sharper used to scan the thyroid (neck area) at least than bands labeled with 32P because the lower- 24 hr after completing each experiment. This energy β radiation does not have the scatter procedure is called a bioassay and is a require- associated with the higher-energy β radiation ment of the institution’s radioactive materials of 32P. The half-life of 33P is also longer (25 license. A very common means of incurring an days compared to 14 days for 32P). Despite internal deposit of 125I during this procedure its higher cost, these features have led some is by the spread of surface contamination with researchers to choose 33P-labeled nucleotides subsequent ingestion or absorption through the for use in experiments such as band/gel shift skin. assays where discrimination of closely spaced gel bands is important. The best way to determine whether addi- MEASURING RADIOACTIVITY There are two main purposes for count- tional shielding is needed when using this iso- ing radioactive materials in the laboratory. The tope is to monitor the source using a β sen- first is to detect the presence and quantity of ra- sitive radiation survey meter. If counts can be dioactive materials in the laboratory as surface detected, add a layer of Plexiglas as described contamination or as an inadequately shielded for 32P. source of radiation for the purposes of safety Working with 125I and regulatory compliance. The second is to determine the presence and/or quantity of the Using 125I to detect immune complexes amount of radioactive label for the purpose of (immunoblots) experimental analysis. 125I that is covalently attached to a molecule such as staphylococcal protein A is not volatile Safety and Regulatory Compliance and therefore is much less hazardous than the Regulations governing the use of radioac- unbound or free form. Most institutions do tive materials in the laboratory address the not insist that work with bound 125I be per- concerns associated with exposure to radia- formed in a hood, but shielding of the γ ra- tion present as surface contamination or as an diation may still be necessary. Lead is a good external radiation field (i.e., exposure at a dis- high-Z material used to shield these γ rays; its tance, typically from an inadequately shielded drawback is its opacity. Commercially avail- radioactive source). Surface contamination is able shields for 125I are made of lead-loaded simply the unintentional presence of radioac- Plexiglas; although heavy, these have the ad- tive material and occurs by the transfer of ac- vantage of being transparent. Alternatively, a tual radioactive material from one surface to piece of lead foil may be taped to a struc- another. To determine the presence of external tural support, although this arrangement does surface contamination, two different method- not provide shielding for the head as a worker ologies are available to the researcher: hand- peers over the lead! held (portable) detectors and liquid scintilla- Incubations of the membrane or blot with tion counters. the [125I] protein A solution and subsequent washes are usually done on a shaker. For shielding during these steps, a piece of lead Hand-held detectors foil may be wrapped around the container. The first method is to use a portable survey Solutions of 125I can be conveniently stored meter, i.e., either a meter with a GM detector for repeated use in a rack placed in a lead or a portable scintillation detector. The choice box. depends on what isotope is being used. GM instruments can be used to detect the pres- ence of the low-energy β emitters 14C, 35S, Using 125I to label proteins or peptides and 33P with an efficiency of detection around in vitro 3% to 5%. For detecting the high-energy β Any experiments that call for the use of emitter 32P, the efficiency of a GM instru- free, unbound 125I should be done behind a ment is ∼30%. As stated previously, a layer of shield in a hood that exhausts the air through Parafilm, commonly used to prevent contam- a charcoal filter (which absorbs the volatile ination of the GM tube in some laboratories, iodine). Most institutions require that such ex- actually prevents the meter from detecting the Safe Use of periments be done in a special hot laboratory low-energy β emitters 14C, 35S, and 33P, and Radioisotopes that has limited access. Since ingested, ab- must be removed prior to use. A.1F.14

Supplement 79 Current Protocols in Molecular Biology The GM detector can also be used to deter- one end to allow the passage of ionizing radia- mine the presence of 125I contamination, al- tion into the tube to initiate the avalanche. The though with much lower efficiency (<1%). tube will implode if the window is accidentally Because of this fact, if very small activity punctured because the pressure inside the tube amounts of 125I are being used (such as pack- is lower than atmospheric pressure. aged in prelabeled kits with <10 µCi), it is best In essence, a single ionizing event inside the to use a portable scintillation detector, which GM detector is amplified so it can be detected. has an efficiency of ∼20% for 125I. As stated previously, the GM counter is not Keep in mind that 3H β radiation has mini- 100% efficient at detecting all isotopes. This mal penetration power and cannot be detected is because a threshold β energy is required to using either a GM or scintillation detector. penetrate the thin window. The β energy from The liquid scintillation counter (see below) is 3H is below this energy threshold. the only readily available means to count 3H, either on a wipe sample or as an analytical Liquid scintillation counters sample. The second method used for determining It is important that meter surveys be per- the presence of radioactive surface contami- formed properly, as one can overlook contam- nation is a wipe test with subsequent count- ination by either moving the meter probe too ing in a liquid scintillation counter. A dry fast or by holding the probe out too far above Whatman filter paper (no. 2 works well) is the surface being monitored. This is especially used to wipe a flat surface, using moderate true for the low-energy β emitters 14C, 35S, and pressure, in the shape of a large “S” ∼6in. 33P. The survey meter probe (GM or scintilla- tall. The wipe is put into a liquid scintilla- tion detector) should be moved slowly, at about tion vial, scintillation fluid (cocktail) is added, 1 to 2 cm/sec, and about 1 cm above the surface and the sample is counted in a scintillation of concern. Any response of the meter higher counter. Any counts greater than background than background must be considered contam- will be indicated on the output of the counter. ination and cleaned up as such. The advantage This method, while not in “real time,” has of a GM survey is the greater area one can the advantage of quantifying the contamina- cover when doing the survey. The downside tion present on the wipe sample, which is not though is that low-level contamination can be easy to do with a GM survey meter. Except overlooked. for tritium, isotopes counted with this method A GM detector works on the principle of the can be detected with >90% efficiency. (Counts Townsend avalanche. A Townsend avalanche can be lost due to an effect termed quenching, occurs when a single gas molecule inside a which is a loss of low-energy events resulting high-voltage electric field becomes ionized, in a shifting of the entire energy spectrum to i.e., loses an electron by the passage of ion- lower energies. This results in a lower counting izing radiation through the gas inside the GM efficiency as compared to unquenched sam- tube. This single electron then acquires enough ples.) Also, this method can detect any isotope kinetic energy traveling across the electric po- used in the laboratory, and can be employed tential to initiate subsequent ionizations in the to find contamination in microcentrifuges and gas, i.e., a cascade, throughout the gas volume. other tight spots where a survey meter can- This creates a large migration of free electrons not fit. However, the down side is that un- to a positively charged anode, which are then less one wipes the surface by pressing directly used to generate an electrical pulse in the elec- on the contamination itself, the contamination trical circuit. The avalanche in the tube is usu- will be overlooked and a false negative can ally stopped by including a quench gas in the occur. tube, which absorbs enough of the free elec- The major advantage of a liquid scintil- tron energy to terminate the discharge. (This lation counter over other radiation detection is a simplified explanation; there is more com- equipment in the laboratory is its ability to plicated physics involved that is not presented detect and count low-energy β radiation with here.) good efficiency. As stated earlier, it is the only In a GM tube, the voltage in the tube can be instrument that can detect tritium (3H). An- 600 to 2000 V and is supplied by batteries and other advantage is its ability to discriminate a voltage regulator in the detector body. The between high- and low-energy β radiation. output of the device is a read-out on a dial, ei- Indeed, samples that contain more than one Standard ther with or without a speaker output. The GM isotope can be counted and the proportion of Measurements, detector tube itself has a very thin window over each separate isotope determined. The device Data, and Abbreviations A.1F.15

Current Protocols in Molecular Biology Supplement 79 is quite complex, and its operation depends local dose rates are possible at near distances on many principles of electronics, electrome- and, because of the long range of 32P β radi- chanics, and physics. ation in air, dose rates even a few feet from Vital to liquid scintillation counting is the the source can be substantial. This radiation use of a scintillation fluid. A scintillation field cannot be measured directly with a GM fluid is a specially formulated liquid com- type of survey instrument, although a rough prised of two kinds of molecules: scintilla- estimate of the dose rate can be made. Using tion molecules and, in much greater quantity, the very common, large area “pancake” type solvent molecules. Together these molecules of GM probe, a meter response to 32P of 2000 serve the purpose of transforming the energy to 3000 cpm indicates an approximate dose of the emitted radiation inside a sample to an rate of 1 mrem per hour. This is a good level energy level that falls in the sensitivity range to aim for when determining the adequacy of of a photomultiplier tube (PMT). The radia- any added shielding used to minimize the dose tion energy in the sample is first transferred to rate from mCi 32P sources. Again, this is only a the solvent, which has an energy level above rough estimate, and Radiation Safety Depart- that of the scintillant and the PMT. The en- ment personnel should be contacted to pro- ergy is then transferred to the scintillant, which vide more accurate determinations of exter- is stimulated to a higher energy level. As the nal dose rates. Ion chamber type instruments scintillant returns to its ground state, it releases are available that can measure actual dose or energy or light that lies within the sensitivity exposure rates more closely than GM survey of the PMT. The light enters the PMT and initi- instruments. ates a cascade down a string of dynodes, where Obviously, the best way to perform a com- the PMT converts a relatively small light sig- plete laboratory survey is to use a combina- nal at one end into a large light pulse at the tion of the two types of surveys (direct meter other. This pulse is then processed electrically survey and wipe test survey) in such a way and counted. as to increase the probability of finding sur- The fact that the sample is in intimate con- face contamination and/or any inadequately tact with the fluid, usually dissolved or in shielded radioactive sources. One can never solution, accounts for the greater sensitivity do too many surveys, and the more experi- of this device over that of the GM detector. ence one gains, the more successful one will The important fact is that higher β energies become at locating and cleaning radioactive in the sample will stimulate greater numbers contamination in the laboratory. of solvent molecules in the first place, lead- ing to a larger number of stimulated scintillant Experimental Analysis molecules. This in turn results in a more in- Due to technological advances with imag- tense event at the start of the dynode string ing machines and computer software, there is and a greater pulse at the other end. This ex- now an increasing number of ways to deter- plains how these detectors can discriminate mine the amount of radioactivity present in β radiation of different energies. In terms of experimental samples. However, if one wants liquid scintillation cocktails (fluors), keep in to know the actual activity (dpm or cpm) of an mind that some chemicals, notably solvents isotope present, the most straightforward way historically used in liquid scintillation cocktail is still to use a scintillation counter to count the formulations, are very difficult and expensive material. While this is often the final stage of to dispose of as radioactive waste, as some an experiment, in that the samples have been are classified as “mixed wastes.” It is best to prepared with this quantitation being the goal, try to avoid these solvents. Some institutions sometimes this measurement is necessary at have restrictions on their use, so the Radiation several points in an experiment to monitor Safety Officer should be contacted for more recovery of the sample. Since low-energy β direction. The best cocktails to use are envi- emitters (3H, 14C, or 35S) require the use of a ronmentally friendly cocktails. scintillation fluor, the portion of sample that is counted is lost in terms of further analysis. External radiation fields: Exposure at a While liquid scintillation counting is more ac- distance curate, 32P samples offer the option of using Radiation present in the form of an external Cerenkov counting, which preserves the entire radiation field (exposure at a distance) is also sample for future types of analysis. Cerenkov possible in the laboratory. This occurs most counting requires a β Emax of at least 0.7 MeV, Safe Use of often when inadequately shielded mCi activ- so 32P samples can be counted with good Radioisotopes ities of 32P are present. In this scenario, high efficiency using this method. For Cerenkov A.1F.16

Supplement 79 Current Protocols in Molecular Biology counting, the sample is simply put into an of cooperation that extends from shielding empty scintillation vial and counted; the with others in mind to helping each other clean counts appearing in the wide window (32Hplus up after accidents occur. 14Cplus32P) are proportional to the amount The specific measures to be taken follow- of 32P present. Keep in mind that Cerenkov ing a spill of radioactive materials naturally counting of a dry sample (a gel band or pro- depend on the type and activity amount of iso- tein pellet, for example) cannot be directly tope involved, the associated chemical or bio- compared with that of a sample counted with logical hazards, and the physical parameters of liquid scintillation fluid because the liquid in the spill (i.e., where and onto what the isotope the sample will act to quench some of the ra- was spilled). However, there are several im- dioactivity. For 125I samples, a γ scintillation mediate steps that should be taken following counter is used; samples can be dry or in liq- any spill. uid form, and no fluor is necessary. Liquid 1. Alert coworkers that there has been scintillation can also be used to count 125I a spill. This will give them the opportunity samples. to protect themselves if need be and to help The advent of phosphor screen technology prepare to clean up the spill. Depending on has enabled researchers to both image and the size and nature of the spill, it will often quantitate gels, blots, or plates by exposing be necessary to notify the Radiation Safety them to a screen and then enabling the in- Office. strument to read the screen. The screens can 2. Restrict movement to and from the site detect 35S, 32P, and 125I with roughly 5-to 10- of the spill. This ensures that radioactive con- fold higher sensitivity than film (depending on tamination is not spread around the laboratory. the isotope) and with a detection range of sam- It is especially important to address those indi- ple variability over 5 orders of magnitude (as viduals who may have come into contact with opposed to 2 orders for film). Computer soft- the radioactive materials. ware is used to select areas of the resultant im- 3. Perform a meter survey on any age and “extract” the amount of radioactivity individuals who may have come in contact therein. The numbers thus generated are of ar- with the radioactive material, including their bitrary units, but can be used for comparative exposed skin, protective clothing, and street purposes, the caveats being that the extracted clothing. If someone’s skin is contaminated, areas must be from the same screen, which first use a portable monitor to identify the spe- itself must be undamaged so that its sensi- cific areas of contamination. Then put that part tivity is uniform. If standards of known ra- of the body under room temperature running dioactive content are used to expose the screen water in a sink. Wash the affected area with alongside the sample, the arbitrary units gen- gentle soap and a soft sponge or washcloth. erated can be translated into real cpm. Several Try to restrict cleaning to the contaminated companies now make such instruments, with area only, so as not to spread the contamina- Fuji, BioRad, and Molecular Dynamics (now tion to other parts of the body. Dry the area, Amersham) being among the best known. The survey, and repeat as necessary or as directed cost of these instruments is still very high; for by Radiation Safety personnel. A shower room this reason many institutions purchase one for may be needed. Contaminated clothing can be use in a core facility. The ability to “cut and carefully removed, placed in a plastic bag, and count” on a computer rather than on real sam- given to Radiation Safety personnel. Contam- ples is highly convenient, and the larger range inated strands of hair can be washed or cut of sensitivity of the phosphor screen eliminates (a new hairstyle may be in order). the need for multiple exposures, as would be 4. Perform an area survey. Useanap- necessary when using film and a densitometer propriate survey meter and work in from the to quantify the signal. supposed outer limits of the spill towards the center. With a nonpermanent marker, outline RESPONDING TO SPILLS the actual hot spots where counts are detected. Despite the best intentions and utmost cau- Do not neglect to survey the sides of walls, tion, accidents happen! Accidents involving cabinets, and equipment close to the spill area, spills of radioactive materials are particularly as radioactive materials may have splashed up insidious because they can be virtually unde- onto these surfaces. tectable if a monitor is not present and turned 5. Clean the area. When attempting to Standard on. For this reason it is best to foster a com- clean any contaminated equipment, floors, Measurements, munity spirit in any laboratory where radioiso- benches, etc., begin by soaking up any visi- Data, and Abbreviations topes are routinely used—specifically, a sense ble radioactive liquid with paper towels and A.1F.17

Current Protocols in Molecular Biology Supplement 79 promptly disposing of the towels in a plastic ACKNOWLEDGEMENTS bag taped to the side of the laboratory bench. Many of the procedures and precautions de- Apply a small amount of decontamination scribed here have evolved over the years and solution to the marked “hot spots” and let it through the millicuries (and are evolving still) set for a few minutes. Then, using three to in the authors’ department (currently Molecu- four paper towels, wipe an area from the outer lar and Cell Biology) at the Salk Institute. The edge to the center of the spill with one swipe authors are indebted to those from whom they of the towels. Immediately discard the paper have learned about the safe use of radioactiv- towels. Repeat this movement with new paper ity in the laboratory. Most of the designs for towels each time, working your way around the shields and other safety equipment shown the spill. Do not reuse any paper towels. This in the figures were created at the Salk Insti- method will minimize the chance of spreading tute in collaboration with Dave Clarkin, Mario contamination to an even greater area. Con- Tengco, and Steve Berry. Safety equipment of tinue this procedure until all the marked areas similar design is available from several com- have been cleaned. mercial vendors, including CBS Scientific and 6. Perform another radiation survey. Research Products International. Survey the entire area to make sure contam- ination has not been overlooked. LITERATURE CITED There is quite a range of commercially Board on Radiation Effects Research (BRER). available foams and sprays made specifically 2006. Health risks from exposure to low to clean radioactive contamination. A dilute levels of ionizing radiation: BEIR VII Phase 2. BRER, National Research Council, solution of phosphoric acid works well to pick The National Academies Press, Washington, 32 up P. Decontamination of centrifuge rotors D.C. Available online at http://www.nap.edu/ can be tricky as their anodized surfaces are books/030909156X/html. sensitive to many detergents; check with the Bursik, S., Meisenhelder J., and Spahn, G. 1999. rotor manufacturer for appropriate cleansing Characterization and minimization of extremity solutions. Many surfaces (particularly metals) doses during 32P metabolic cell labeling. Health prove resistant to even herculean cleaning ef- Phys. 77:595-600. forts; in these instances the best that can be Klein, R., Reginatto, M., Party, E., and Gershey, E. done is to remove all contamination possi- 1990. Practical radiation shielding for biomedi- cal research. Radiat. Prot. Manage. 7:30-37. ble and then shield whatever remains until the radioactivity decays to minimal levels. Lederer, C.M., Hollander, J.M., and Perlman, I. (eds.) 1967. Table of Radioisotopes, 6th ed. John Wiley & Sons, New York. CONCLUSION Meisenhelder, J. and Hunter, T. 1988. Radioactive When working with radioisotopes, it is best protein-labeling techniques. Nature 335:120. to plan ahead and then plan ahead some more. Shleien, B. (ed.) 1987. Radiation Safety Manual for The more thoroughly familiar a researcher be- Users of Radioisotopes in Research and Aca- comes with all aspects of their work—by be- demic Institutions. Nucleon Lectern Associates, ing proactive and addressing all questions and Olney, Md. concerns beforehand (the science, the equip- ment, and the technique)—the more success- INTERNET RESOURCES ful the researcher will be at providing for their http://web.princeton.edu/sites/ehs own safety and the safety of those working Princeton University Environmental Health and around them. Safety Web site containing radioisotope fact sheets.

Safe Use of Radioisotopes A.1F.18

Supplement 79 Current Protocols in Molecular Biology Centrifuges and Rotors APPENDIX 1G

Centrifugation runs described in this book usually specify a relative centrifugal force (RCF; measured in × g), corresponding to a speed (in rpm) for a particular centrifuge and rotor model. As available equipment will vary from laboratory to laboratory, the investi- gator must be able to adapt these specifications to other centrifuges and rotors.

The relationship between RCF and speed (rpm) is determined by the following equation: 2 RCF =1.12r (rpm ⁄ 1000) where r is the rotating radius between the particle being centrifuged and the axis of rotation. In most cases, an accurate conversion from speed to relative centrifugal force

(or vice versa) can be obtained using the maximum value of r—or rmax—equal to the distance between the axis of rotation and the bottom of the centrifuge tube as it sits in the well or bucket of the rotor.

Table A.1G.1 provides rmax values for commonly used rotors manufactured by Du Pont (Sorvall), Beckman, Fisher, and IEC. There are situations (e.g., where an adapter is used to fit a smaller tube into a larger rotor well) where rmax will not accurately represent the effective rotating radius. In such cases, the manual for the rotor should be consulted to obtain the appropriate value of r. As an alternative to use of the above equation, the nomograms in Figures A.1G.1 and

A.1G.2 make it possible to determine the RCF where speed and rmax are known, or the speed where RCF and rmax are known. This is done by aligning a ruler across the two known values and reading the unknown value at the point where the ruler crosses the remaining column. Figure A.1G.1 should be used for centrifuge runs <21,000 rpm, while Figure A.1G.2 should be used for faster spins.

NOTE: In this manual, for spins involving microcentrifuges built to the Eppendorf standard, a shortened style of reference including only the speed (in rpm) is used. All of these instruments have approximately the same rotating radius; hence the same speed will yield the same RCF value from machine to machine. Microcentrifuge spins may also be described as at “top speed” or “maximum speed,” meaning 12,000 to 14,000 rpm, which is the maximum speed for all Eppendorf-type microcentrifuges.

CAUTION: Do not exceed maximum rotor speed! For Beckman ultracentrifuges, the maximum speed for each rotor is denoted by its name, e.g., the maximum speed of the Beckman VTi 80 rotor is 80,000 rpm. This speed refers only to centrifugation of solutions below a particular allowed density, which differs among rotors (see user manual). For centrifugation of high-density solutions, rotor maximum speed can be determined as: 1 ⁄2 reduced rpm = rpmmax(A/B) , where A=allowed density and B=density of solution. A=1.7 g/ml for several vertical rotors (including VTi 80 and VTi 50), and 1.2 g/ml for several swinging-bucket rotors (including SW 55 Ti, 28, 28.1, 40 Ti, 50.1). For gradients using heavy salts such as CsCl, particularly at low temperatures, maximum rpm should be reduced to prevent precipitation (see user manual). Table A.1G.2 describes centrifuge tube materials and their properties, including optical properties, appropriate methods for sterilization, and chemical resistances (tolerance to various media, organic solvents, and alcohols). Standard Measurements, Data, and Abbreviations Current Protocols in Molecular Biology (1996) A.1G.1-A.1G.6 A.1G.1 Copyright © 2000 by John Wiley & Sons, Inc. Supplement 35 180 90,000 21,000 20,000

50,000 150 15,000

10,000 10,000

100

5000 90

80 5000

70 1000

60

500

50

200 2000

40 100

50 40 30 30 1000

20 25 15.7 750 Rotating radius Relative centrifugal Rotor speed (mm) force (x g) (rpm)

Figure A.1G.1 Nomogram for conversion of relative centrifugal force to rotor speed in low-speed centrifuge runs. To determine an unknown value in a given column, align ruler through known values in other two columns. Desired value is found at the intersection of the ruler with the column of interest. For faster centrifugations, use Figure A.1G.2. A more precise conversion can be obtained using the equation at the beginning of this appendix. See Table A.1G.1 for rotating radii of commonly used rotors.

Centrifuges and Rotors A.1G.2

Supplement 35 Current Protocols in Molecular Biology 80,000 75,000 70,000

65,000

60,000

1,000,000 900,000 800,000 700,000 50,000 600,000 200 500,000 180 400,000 160 300,000 40,000 140 200,000 120

100 90 100,000 90,000 30,000 80 80,000 70,000 70 60,000 60 50,000 40,000

50 30,000

40 20,000 20,000

30 10,000 9000 8000 7000 6000 20 5000 4000

3000

2000

10 10,000 1000 Rotating radius Relative centrifugal Rotor speed (mm) force (x g) (rpm)

Figure A.1G.2 Nomogram for conversion of relative centrifugal force to rotor speed in high-speed centrifuge runs. For slower centrifugations and instructions for using the nomogram, use Figure A.1G.1. A more precise conversion can be obtained using the equation at the beginning of this appendix. See Table A.1G.1 for rotating radii of commonly used rotors. A.1G.3

Current Protocols in Molecular Biology Supplement 35 Table A.1G.1 Maximum Rotating Radii for Common Rotors, Grouped by Centrifuge Model

a a Rotor model rmax (mm) Rotor model rmax (mm) For Sorvall centrifuge models GLC-1, GH-3.7 (buckets) 204 GLC-2, GLC-2B, GLC-3, GLC-4, GH-3.7 (microplate carrier) 168 RT-6000B, T-6000, T-6000B GH-3.8 (buckets) 204 A/S400 140 GH-3.8 (microplate carrier) 168 H-1000B 186 For Beckman TJ-6 series centrifuges HL-4 with 50-ml bucket 180 TA-10 123 HL-4 with 100-ml bucket 204 TA-24 108 HL-4 with Omni-Carrier 163 TA-24 with adapter for 123 M and A-384 (inner row) 91 10-ml tubes M and A-384 (outer row) 121 TH-4 (stainless steel 186 SP/X and A-500 (inner row) 82 buckets) SP/X and A-500 (outer row) 123 TH-4 (100-ml tube holders) 201 For Sorvall centrifuge models RC-3, TH-4 (microplate carrier) 165 RC-3B, RC-3C) For Beckman AccuSpin H-2000B 261 AA-10 123 H-4000 and HG-4L 230 AA-24 108 H-6000A 260 AA-24 with adapter for 123 HL-8 with Omni-Carrier 221 10-ml tubes HL-8 with 50-ml bucket 238 AH-4 163 HL-8 with 100-ml bucket 247 For Beckman J6 series centrifuges HL-2 and HL-2B 166 JR-3.2 206 LA/S400 140 JS-2.9 265 For Sorvall centrifuge models RC-2, JS-3.0 254 RC-2B, RC-5, RC-5B, RC-5C JS-4.0 226 GSA 145 JS-4.2 254 GS-3 151 JS-4.2SM 248 HB-4 147 JS-5.2 226 HS-4 with 250-ml bucket 172 Microplate carrier 214 SA-600 129 (6-bucket rotors) SE-12 93 Microplate carrier 192 SH-80 101 (4-bucket rotors) SM-24 (inner row) 91 For Beckman J2-21 series centrifuges SM-24 (outer row) 110 JA-10 158 SS-34 107 JA-14 137 SV-80 101 JA-17 123 SV-288 90 JA-18 132 TZ-28 95 JA-18.1 (25° angle) 112 For Sorvall ultracentrifuges JA-18.1 (45° angle) 116 T-865 91 JA-20 108 T-865.1 87.1 JA-20.1 115 T-875 87.1 JA-21 102 T-880 84.7 JCF-Z 89 T-1270 82 JCF-Z with small pellet core 81 TFT-80.2 65.5 JE-6B 125 TFT-80.4 60.1 JS-7.5 165 For Beckman GP series centrifuges JS-13 142 GA-10 123 JS-13.1 140 GA-24 123 JV-20 93 GA-24 with adapter for 108 10-ml tubes continued Centrifuges and Rotors A.1G.4

Supplement 35 Current Protocols in Molecular Biology Table A.1G.1 Maximum Rotating Radii for Common Rotors, Grouped by Centrifuge Model, continued

a a Rotor model rmax (mm) Rotor model rmax (mm) For Beckman series L7 and L8 Type 80 Ti 84.0 ultracentrifuges VAC 50 86.4 SW 25.1 129.2 VC 53 78.8 SW 28 161.0 VTi 50 86.6 SW 28.1 171.3 VTi 65 85.4 SW 30 123.0 VTi 65.2 87.9 SW 30.1 123.0 VTi 80 71.1 SW 40 Ti 158.8 For Beckman Airfuge ultracentrifuge SW 41 Ti 153.1 A-95 17.6 SW 50.1 107.3 A-100/18 14.6 SW 55 Ti 108.5 A-100/30 16.5 SW 60 Ti 120.3 A-110 14.7 SW 65 Ti 89.0 ACR-90 (2.4-ml liner) 11.8 Type 15 142.1 ACR-90 (3.5-ml liner) 13.4 Type 19 133.4 Batch rotor 14.6 Type 21 121.5 EM-90 13.0 Type 25 100.4 For Beckman TL-100 series ultracentrifuges Type 30 104.8 TLA-100 38.9 Type 30.2 94.2 TLA 100.1 38.9 Type 35 104.0 TLA-100.2 38.9 Type 40 80.8 TLA-100.3 48.3 Type 40.3 79.5 TLA-45 55.1 Type 42.1 98.6 TLS-55 76.4 Type 42.2 Ti 104 TLV-100 35.7 Type 45 Ti 103 Type 50 70.1 Miscellaneous centrifuges and rotorsb Type 50 Ti 80.8 Clay Adams Dynac —c Type 50.2 Ti 107.9 Fisher Centrific 113 Type 50.3 Ti 79.5 Fisher Marathon 21K with 160 Type 50.4 Ti (inner row) 96.4 4-place rotor Type 50.4 Ti (outer row) 111.4 IEC Clinical centrifuge with 155 Type 55.2 Ti 100.3 4-place swinging-bucket Type 60 Ti 89.9 rotor c Type 65 77.7 IEC general-purpose — centifuge models HN, Type 70 Ti 91.9 HN-SII, and Centra-4 Type 70.1 Ti 82.0 Type 75 Ti 79.7

aSorvall centrifuges and rotors are a product of Du Pont Company Medical Products, Beckman centrifuges are a product of Beckman Instruments, IEC centrifuges are a product of International Equipment Co., Clay Adams Dynac centrifuges are a product of Becton Dickinson Labware, and Fisher centrifuges are a product of Fisher Scientific. For ordering information see APPENDIX4 . bThese instruments are often loosely referred to as “clinical,” “tabletop,” or “low-speed” centrifuges. cThese instruments accept a wide range of trunnion-ring rotors with variable rotating radii, as well as fixed-angle and swinging-bucket rotors that in turn accept a variety of adapters making it possible to spin different numbers tubes of various sizes. For instance, the commonly used IEC 958 trunnion-ring rotor may be adjusted to a radii ranging from 137 to 181 mm, depending on the trunnion-ring chosen. It is therefore necessary to consult the manual for the specific system being used to obtain an accurate speed to RCF conversion.

Standard Measurements, Data, and Abbreviations A.1G.5

Current Protocols in Molecular Biology Supplement 35 Table A.1G.2 Centrifuge Tube Materials and Their Propertiesa

Supplement 35 Supplement Optical Type Puncturable Sliceable Reusable Sterilization methods Chemical resistancesb A.1G.6 property Ultra-Clear thin-walled Transparent Yes Yes Cold sterilization only, but not Good tolerance to all gradient media except Standard tubes Yes with alcohol alkaline ones (>pH 8). Satisfactory for most weak nnnn Quick-Seal tubes No acids and a few weak bases. Unsatisfactory for DMSO and most organic solvents, including all alcohols. Polyallomer thin-walled Translucent Yes Yes Can be autoclaved on a test tube Good tolerance to all gradient media, including Standard tubes Yes rack at 121°C alkaline ones. Satisfactory for most acids, many Quick-Seal tubes No bases, many alcohols, DMSO, and some organic solvents. Polyallomer thick-walled Translucent Can be autoclaved on a test tube Good tolerance to all gradient media, including Tubes No No Yes rack at 121°C alkaline ones. Satisfactory for most acids, many Bottles No No Yes bases, many alcohols, DMSO, and some organic solvents. Polycarbonate thick-walled Transparent No No Cold sterilization recommended, Good tolerance to all gradient media except Tubes Yes but not with alcohol. Can be alkaline ones (>pH 8). Satisfactory for some weak Bottles Yes autoclaved at 121°C, but tube acids. Unsatisfactory for all bases, alcohols, and life may be reduced. other organic solvents. Cellulose propionate tubes Transparent No No Yes Cold sterilization only, but not Good tolerance to all gradient media, including with alcohol alkaline ones. Unsatisfactory for most acids, bases, alcohols, and other organic solvents. Polypropylene Translucent No No Can be autoclaved at 121°C Good tolerance to all gradient media, including Tubes Yes alkaline ones. Satisfactory for many acids, bases, Bottles Yes and alcohols. Unsatisfactory for most organic solvents. Stainless steel tubes Opaque No No Yes Can be autoclaved. Dry Good tolerance to many organic solvents. thoroughly before storage. Marginal with many gradient media and salts. Unsatisfactory for most acids and many bases. Current Protocols in Molecular Biology Molecular in Protocols Current Polyethylene tubes Translucent No No Yes Can be autoclaved at 121°C Good tolerance to a wide range of chemicals. Suitable for use with strong acids and bases. Unsatisfactory for most organic solvents. Corex/Pyrex Transparent No No Can be autoclaved at 121°C Good tolerance to a wide range of gradient media. Tubes Yes Corex has greater resistance to bases and acids. Bottles Yes aTable reproduced by permission of Beckman Instruments. bChemical resistances are described here in general terms, and are not meant by Beckman Instruments or John Wiley & Sons to express or imply any guarantee of safety based on these recommendations or resistances. If there is any doubt about a particular solution, it should be tested under actual operating conditions to evaluate the performance of a tube material. For more detailed information regarding specific media and solvents, consult Beckman Instruments. High-vapor-pressure inflammable solvents should not be handled in close vicinity to centrifuges because of possible ignition by sparking switches, relay contacts, or motor brushes. Safe Use of Hazardous Chemicals APPENDIX 1H Carrying out the protocols in this manual may result in exposure to toxic chemicals or carcinogenic, mutagenic, or teratogenic reagents (see Table A.1H.1). Cautionary notes and some specific guidelines are included in many instances throughout the manual; however, users must proceed with the prudence and precautions associated with good laboratory practice, under the supervision of those responsible for implementing lab safety programs at their institutions. It is not possible in the space available to list all the precautions required for handling hazardous chemicals. Many texts have been written about laboratory safety (see Literature Cited and Key References). Obviously, all national and local laws should be obeyed, as well as all institutional regulations. Controlled substances are regulated by the Drug Enforcement Administration (http://www.doj.gov/dea). By law, Material Safety Data Sheets (MSDSs) must be readily available. All laboratories should have a Chemical Hygiene Plan (29CFR Part 1910.1450); institutional safety officers should be consulted as to its implementation. Help is (or should be) available from your institutional Safety Office; use it. Chemicals must be stored properly for safety. Certain chemicals cannot be easily or safely mixed with and should not be stored near certain other chemicals, because their reaction is violently exothermic or yields a toxic product. Some examples of incompatibility are listed in Table A.1H.2. When in doubt, always consult a current MSDS for information on reactivity, handling, and storage. Chemicals should be separated into general hazard classes and stored appropriately. For example, flammable chemicals such as alcohols, ketones, aliphatic and aromatic hydrocarbons, and other materials labeled flammable should be stored in approved flammable storage cabinets, with those also requiring refrigeration being kept in explosion-proof refrigerators. Strong oxidizers must be segre- gated. Strong acids (e.g., sulfuric, hydrochloric, nitric, perchloric, and hydrofluoric) should be stored in a separate cabinet well removed from strong bases and from flammable organics. An exception is glacial acetic acid, which is both corrosive and flammable, and which must be stored with the flammables. Facilities should be appropriate for working with hazardous chemicals. In particular, hazardous chemicals should be handled only in chemical fume hoods, not in laminar flow cabinets. The functioning of the fume hoods should be checked periodically. Laboratories should also be equipped with safety showers and eye-wash facilities. Again, this equip- ment should be tested periodically to ensure that it functions correctly. Other safety equipment may be required depending on the nature of the materials being handled. In addition, researchers should be trained in the proper procedures for handling hazardous chemicals as well as other laboratory operations—e.g., handling of compressed gases, use of cryogenic liquids, operation of high-voltage power supplies, and operation of lasers of all types. Before starting work, know the physical and chemical hazards of the reagents used. Wear appropriate protective clothing and have a plan for dealing with spills or accidents; coming up with a good plan on the spur of the moment is very difficult. For example, have the appropriate decontaminating or neutralizing agents prepared and close at hand. Small spills can probably be cleaned up by the researcher. In the case of larger spills, the area should be evacuated and help should be sought from those experienced in and equipped for dealing with spills—e.g., the institutional Safety Office. Protective equipment should include, at a minimum, eye protection, a lab coat, and gloves. In certain circumstances other items of protective equipment may be necessary (e.g., a Standard Measurements, face shield). Different types of gloves exhibit different resistance properties (Table Data, and Abbreviations

Contributed by George Lunn and Gretchen Lawler A.1H.1 Current Protocols in Molecular Biology (2002) A.1H.1-A.1H.33 Copyright © 2002 by John Wiley & Sons, Inc. Supplement 58 Table A.1H.1 Commonly Used Hazardous Chemicalsa

Chemical Hazards Remarksb Acetic acid, glacial Corrosive, flammable liquid Acetonitrile Flammable liquid, teratogenic, toxic Acridine orange Carcinogenic, mutagenic See Basic Protocol 2 Acrylamide Carcinogenic, toxic Use dust mask; polyacrylamide gels contain residual acrylamide monomer and should be handled with gloves; acrylamide may polymerize with violence on melting at 86°C Alcian blue 8GX See Basic Protocol 2 Alizarin red S (monohydrate) p-Amidinophenylmethanesulfonyl Enzyme inhibitor See Basic Protocol 11 fluoride (APMSF) 7-Aminoactinomycin D (7-AAD) Carcinogenic 4-(2-Aminoethyl)benzenesulfonyl Mutagenic, enzyme inhibitor See Basic Protocol 11 fluoride (AEBSF) Ammonium hydroxide, concentrated Corrosive, lachrymatory, toxic Azure A Mutagenic See Basic Protocol 2 Azure B Mutagenic See Basic Protocol 2 Benzidine (BDB) Carcinogenic, toxic See Basic Protocol 1 Bisacrylamide Toxic Boron dipyrromethane derivatives Toxic (BODIPY dyes) Brilliant blue R Carcinogenic, mutagenic See Basic Protocol 2 5-Bromodeoxyuridine (BrdU) Mutagenic, teratogenic, photosensitizing Cetylpyridinium chloride (CPC) Toxic Cetyltrimethylammonium bromide (CTAB) Corrosive, teratogenic, toxic Chloroform Carcinogenic, teratogenic, toxic Chlorotrimethylsilane Carcinogenic, corrosive, flammable Reacts violently with water; liquid, toxic see Basic Protocol 3 Chromic/sulfuric acid cleaning solution Carcinogenic, corrosive, oxidizer, Replace with suitable toxic commercially available cleanser Chromomycin A3 (CA3) Teratogenic, toxic Congo red Mutagenic, teratogenic See Basic Protocol 2 Coomassie brilliant blue G Mutagenic See Basic Protocol 2 Crystal violet See Basic Protocol 2 Cresyl violet acetate Mutagenic See Basic Protocol 2 Cyanides (e.g., KCN, NaCN) Toxic Contact with acid will liberate HCN gas; see Basic Protocol 4 Cyanines (e.g., Cy3, Cy5) Toxic Cyanogen bromide (CNBr) Toxic See Basic Protocol 4

continued

A.1H.2

Supplement 58 Current Protocols in Molecular Biology Table A.1H.1 Commonly Used Hazardous Chemicalsa, continued

Chemical Hazards Remarksb

2′-Deoxycoformycin (dCF, pentostatin) Teratogenic, toxic 4′,6-Diamidino-2-phenylindole (DAPI) Mutagenic Diaminobenzidine (DAB) Carcinogenic See Basic Protocol 1 1,4-Diazabicyclo[2,2,2]-octane (DABCO) Toxic Forms an explosive complex with hydrogen peroxide Dichloroacetic acid (DCA) Carcinogenic, corrosive, toxic Dichloromethane (methylene chloride) Carcinogenic, mutagenic, teratogenic, toxic Diethylamine (DEA) Corrosive, flammable liquid, toxic Diethylpyrocarbonate (DEPC) Carcinogenic, toxic Diethyl sulfate Carcinogenic, teratogenic, toxic See Basic Protocol 5 Diisopropyl fluorophosphate (DFP) Highly toxic, cholinesterase inhibitor, See Basic Protocol 11 neurotoxin Dimethyldichlorosilane Corrosive, flammable liquid, toxic See Basic Protocol 3 Dimethyl sulfate (DMS) Carcinogenic, toxic See Basic Protocol 5 Dimethyl sulfoxide (DMSO) Flammable liquid, toxic Enhances absorption through skin Diphenylamine (DPA) Teratogenic, toxic 2,5-Diphenyloxazole (PPO) Toxic Dithiothreitol (DTT) Toxic Eosin B See Basic Protocol 2 Erythrosin B Carcinogenic, mutagenic See Basic Protocol 2 Ether Flammable liquid, toxic May form explosive peroxides on standing; do not dry with NaOH or KOH Ethidium bromide (EB) Mutagenic, toxic See Basic Protocol 2 or 6 Ethyl methanesulfonate (EMS) Carcinogenic, toxic See Basic Protocol 5 Fluorescein and derivatives Carcinogenic, toxic 5-Fluoro-2′-deoxyuridine (FUdR) Teratogenic, toxic Fluoroorotic acid (FOA) Toxic Formaldehyde Carcinogenic, flammable liquid, teratogenic, toxic Formamide Teratogenic, toxic Formic acid Corrosive, toxic May explode when heated >180°C in a sealed tube Glutaraldehyde Corrosive, teratogenic, toxic Guanidinium thiocyanate Toxic Hoechst 33258 dye Mutagenic, toxic Hydrochloric acid, concentrated Corrosive, teratogenic, toxic

continued

Standard Measurements, Data, and Abbreviations A.1H.3

Current Protocols in Molecular Biology Supplement 58 Table A.1H.1 Commonly Used Hazardous Chemicalsa, continued

Chemical Hazards Remarksb

Hydrogen peroxide (30%) Carcinogenic, corrosive, mutagenic, Avoid bringing into contact oxidizer with organic materials, which may form explosive peroxides; may decompose violently in contact with metals, salts, or oxidizable materials; see Basic Protocol 7 Hydroxylamine Corrosive, flammable, mutagenic, Explodes in air at >70°C toxic 3-β-Indoleacrylic acid (IAA) Carcinogenic Iodine Corrosive, toxic See Basic Protocol 8 Iodoacetamide Carcinogenic, mutagenic, toxic Janus green B Carcinogenic, mutagenic See Basic Protocol 2 Lead compounds Carcinogenic, toxic 2-Mercaptoethanol (2-ME) Stench, toxic Mercury compounds Teratogenic, toxic See Basic Protocol 9 Methionine sulfoximine (MSX) Teratogenic, toxic Methotrexate (amethopterin) Carcinogenic, mutagenic, teratogenic, toxic Methylene blue Mutagenic, toxic See Basic Protocol 2 Methyl methanesulfonate (MMS) Carcinogenic, toxic See Basic Protocol 5 Mycophenolic acid (MPA) Teratogenic, toxic Neutral red Mutagenic See Basic Protocol 2 Nigrosin, water soluble See Basic Protocol 2 Nitric acid, concentrated Corrosive, oxidizer, teratogenic, toxic Nitroblue tetrazolium (NBT) Toxic Orcein, synthetic See Basic Protocol 2 Oxonols Toxic Paraformaldehyde Toxic Phenol Carcinogenic, corrosive, teratogenic, Readily absorbed through toxic the skin Phenylmethylsulfonyl fluoride (PMSF) Enzyme inhibitor See Basic Protocol 11 Phorbol 12-myristate 13-acetate (PMA) Carcinogenic, toxic Phycoerythrins (PE) Toxic Piperidine Flammable liquid, teratogenic, toxic Potassium hydroxide, concentrated Corrosive, toxic Produces a highly exothermic reaction when solid is added to water Propane sultone Carcinogenic, toxic See Basic Protocol 5 Propidium iodide (PI) Mutagenic See Basic Protocol 2 or 6 Pyridine Flammable liquid, toxic Rhodamine and derivatives Toxic Rose Bengal Carcinogenic, teratogenic See Basic Protocol 2 Safranine O Mutagenic See Basic Protocol 2

continued

A.1H.4

Supplement 58 Current Protocols in Molecular Biology Table A.1H.1 Commonly Used Hazardous Chemicalsa, continued

Chemical Hazards Remarksb

Sodium azide Carcinogenic, toxic Adding acid liberates explosive volatile, toxic hydrazoic acid; can form explosive heavy metal azides, e.g., with plumbing fixtures—do not discharge down drain; see Basic Protocol 10 Sodium deoxycholate (Na-DOC) Carcinogenic, teratogenic, toxic Sodium dodecyl sulfate (sodium lauryl sulfate, Sensitizing, toxic SDS) Sodium hydroxide, concentrated Corrosive, toxic A highly exothermic reaction ensues when the solid is added to water Sodium nitrite Carcinogenic Sulfuric acid, concentrated Corrosive, oxidizer, teratogenic, toxic Reaction with water is very exothermic; always add concentrated sulfuric acid to water, never water to acid SYTO dyes Toxic Tetramethylammonium chloride (TMAC) Toxic N,N,N′,N′-Tetramethyl-ethylenediamine Corrosive, flammable liquid, toxic (TEMED) Texas Red (sulforhodamine 101, acid chloride) Toxic Toluene Flammable liquid, teratogenic, toxic Toluidine blue O Mutagenic, toxic See Basic Protocol 2 Nα-p-Tosyl-L-lysine chloromethyl Toxic, enzyme inhibitor See Basic Protocol 11 ketone (TLCK) N-p-Tosyl-L-phenylalanine chloromethyl ketone Toxic, mutagenic, enzyme inhibitor See Basic Protocol 11 (TPCK) Trichloroacetic acid (TCA) Carcinogenic, corrosive, teratogenic, toxic Triethanolamine acetate (TEA) Carcinogenic, toxic Trifluoroacetic acid (TFA) Corrosive, toxic Trimethyl phosphate (TMP) Carcinogenic, mutagenic, teratogenic May explode on distillation Trypan blue Carcinogenic, mutagenic, teratogenic See Basic Protocol 2 Xylenes Flammable liquid, teratogenic, toxic aFor extensive information on the hazards of these and other chemicals as well as cautionary details, see Bretherick (1986), O’Neil (2001), Furr (2000), Lewis (1999), Lunn and Sansone (1994a), and Bretherick et al. (1999). bCAUTION: These chemicals should be handled only in a chemical fume hood by knowledgeable workers equipped with eye protection, lab coat, and gloves. The laboratory should be equipped with a safety shower and eye wash. Additional protective equipment may be required.

A.1H.3). No gloves resist all chemicals, and no gloves resist any chemicals indefinitely. Disposable gloves labeled “exam” or “examination” are primarily for protection from biological materials (e.g., viruses, bacteria, feces, blood). They are not designed for and usually have not been tested for resistance to chemicals. Disposable gloves generally offer extremely marginal protection from chemical hazards in most cases and should be Standard removed immediately upon contamination before the chemical can pass through. If Measurements, possible, design handling procedures to eliminate or reduce potential for contamination. Data, and Never assume that disposable gloves will offer the same protection or even have the same Abbreviations A.1H.5

Current Protocols in Molecular Biology Supplement 58 Table A.1H.2 Examples of Chemical Incompatibility

Chemical Incompatible with Acetic acid Aldehydes, bases, carbonates, chromic acid, ethylene glycol, hydroxides, hydroxyl compounds, metals, nitric acid, oxidizers, perchloric acid, peroxides, phosphates, permanganates, xylene Acetone Acids, amines, concentrated nitric and sulfuric acid mixtures, oxidizers, plastics Acetylene Copper, halogens, mercury, oxidizers, potassium, silver Alkali metals, alkaline earth Acids, aldehydes, carbon dioxide, carbon tetrachloride or metals other chlorinated hydrocarbons, halogens, ketones, plastics, sulfur, water Ammonia (anhydrous) Acids, aldehydes, amides, calcium hypochlorite, hydrofluoric acid, halogens, heavy metals, mercury, oxidizers, plastics, sulfur Ammonium nitrate Acids, alkalis, chlorates, chloride salts, flammable and combustible materials, metals, organic materials, phosphorus, reducing agents, sulfur, urea Aniline Acids, aluminum, dibenzoyl peroxide, oxidizers, plastics Arsenical materials Any reducing agent Azides Acids, heavy metals, oxidizers Bromine Acetaldehyde, alcohols, alkalis, amines, ammonia, combustible materials, ethylene, fluorine, hydrogen, ketones (e.g., acetone, carbonyls), metals, petroleum gases, sodium carbide, sulfur Calcium oxide Acids, ethanol, fluorine, organic materials, water Carbon (activated) Alkali metals, calcium hypochlorite, halogens, oxidizers Carbon tetrachloride Sodium Chlorates Acids, ammonium salts, finely divided organic or combustible materials, powdered metals, sulfur Chlorine Acetylene or other hydrocarbons, alcohols, ammonia, benzene, butadiene, butane, combustible materials, ethylene, flammable compounds (e.g., hydrazine), hydrogen, hydrogen peroxide, iodine, metals, methane, nitrogen, oxygen, propane (or other petroleum gases), sodium carbide, sodium hydroxide Chlorine dioxide Ammonia, hydrogen, hydrogen sulfide, mercury, methane, organic materials, phosphine, phosphorus, potassium hydroxide, sulfur Chromic acid, chromic Acetic acid, acetone, alcohols, alkalis, ammonia, bases, oxide benzene, camphor, flammable liquids, glycerin (glycerol), hydrocarbons, metals, naphthalene, organic materials, phosphorus, plastics Copper Acetylene, calcium, hydrocarbons, hydrogen peroxide, oxidizers Cumene hydroperoxide Acids (organic or inorganic) Cyanides Acids, alkaloids, aluminum, iodine, oxidizers, strong bases Flammable liquids Ammonium nitrate, chromic acid, halogens, hydrogen peroxide, nitric acid, oxidizing agents in general, oxygen, sodium peroxide Fluorine All other chemicals

continued Safe Use of Hazardous Chemicals A.1H.6

Supplement 58 Current Protocols in Molecular Biology Table A.1H.2 Examples of Chemical Incompatibility, continued

Chemical Incompatible with

Hydrocarbons See flammable liquids (liquid or gas) Hydrocyanic acid Alkali, nitric acid Hydrofluoric acid Ammonia, metals, organic materials, plastics, silica (glass, including fiberglass), sodium Hydrogen peroxide All organics, most metals or their salts, nitric acid, phosphorus, sodium, sulfuric acid Hydrogen sulfide Acetylaldehyde, fuming nitric acid, metals, oxidizers, sodium, strong bases Hydroperoxide Reducing agents Hypochlorites Acids, activated carbon Iodine Acetylaldehyde, acetylene, ammonia, hydrogen, metals, sodium Mercury Acetylene, aluminum, amines, ammonia, calcium, fulminic acid, lithium, oxidizers, sodium Nitric acid Acids, nitrites, metals, most organics, plastics, sodium, sulfur, sulfuric acid Nitrites Acids Nitroparaffins Amines, inorganic bases Oxalic acid Mercury, oxidizers, silver, sodium chlorite Oxygen All flammable and combustible materials, ammonia, carbon monoxide, grease, metals, oil, phosphorus, polymers Perchloric acid All organics, bismuth and alloys, dehydrating agents, grease, hydrogen halides, iodides, paper, wood Peroxides, organic Acids (organic or mineral), avoid friction, store cold Phosphorus (white) Air, alkalis, oxygen, reducing agents Potassium chlorate Acids, ammonia, combustible materials, fluorine, hydrocarbons, metals, organic materials, reducing agents, sugars Potassium perchlorate Alcohols, combustible materials, fluorine, hydrazine, metals, organic matter, reducing agents, sulfuric acid Potassium permanganate Benzaldehyde, ethylene glycol, glycerin, sulfuric acid Selenides and tellurides Reducing agents Silver Acetylene, ammonium compounds, fulminic acid, oxalic acid, ozonides, peroxyformic acid, tartaric acid Sodium Acids, carbon dioxide, carbon tetrachloride, hydrazine, metals, oxidizers, water Sodium nitrate Acetic anhydride, acids, metals, organic matter, peroxyformic acid, reducing agents Sodium peroxide Acetic anhydride, benzaldehyde, benzene, carbon disulfide, ethyl acetate, ethyl or methyl alcohol, ethylene glycol, furfural, glacial acetic acid, glycerin, hydrogen sulfide, metals, methyl acetate, oxidizers, peroxyformic acid, phosphorus, reducing agents, sugars, water Sulfides Acids Sulfuric acid Alcohols, bases, chlorates, perchlorates, permanganates of potassium, lithium, sodium, magnesium, calcium

Standard Measurements, Data, and Abbreviations A.1H.7

Current Protocols in Molecular Biology Supplement 58 Table A.1H.3 Chemical Resistance of Commonly Used Glovesa,b

Chemical Neoprene gloves Latex gloves Butyl gloves Nitrile gloves *Acetaldehyde VG G VG G Acetic acid VG VG VG VG *Acetone G VG VG P Ammonium hydroxide VG VG VG VG *Amyl acetate F P F P Aniline G F F P *Benzaldehyde F F G G *Benzene P P P F Butyl acetate G F F P Butyl alcohol VG VG VG VG Carbon disulfide F F F F *Carbon tetrachloride F P P G *Chlorobenzene F P F P *Chloroform G P P E Chloronaphthalene F P F F Chromic acid (50%) F P F F Cyclohexanol G F G VG *Dibutyl phthalate G P G G Diisobutyl ketone P F G P Dimethylformamide F F G G Dioctyl phthalate G P F VG Epoxy resins, dry VG VG VG VG *Ethyl acetate G F G F Ethyl alcohol VG VG VG VG *Ethyl ether VG G VG G *Ethylene dichloride F P F P Ethylene glycol VG VG VG VG Formaldehyde VG VG VG VG Formic acid VG VG VG VG Freon 11, 12, 21, 22 G P F G *Furfural G G G G Glycerin VG VG VG VG Hexane F P P G Hydrochloric acid VG G G G Hydrofluoric acid (48%) VG G G G Hydrogen peroxide (30%) G G G G Ketones G VG VG P Lactic acid (85%) VG VG VG VG Linseed oil VG P F VG Methyl alcohol VG VG VG VG Methylamine F F G G Methyl bromide G F G F *Methyl ethyl ketone G G VG P *Methyl isobutylketone F F VG P Methyl methacrylate G G VG F Monoethanolamine VG G VG VG

continued Safe Use of Hazardous Chemicals A.1H.8

Supplement 58 Current Protocols in Molecular Biology Table A.1H.3 Chemical Resistance of Commonly Used Glovesa,b, continued

Chemical Neoprene gloves Latex gloves Butyl gloves Nitrile gloves Morpholine VG VG VG G Naphthalene G F F G Naphthas, aliphatic VG F F VG Naphthas, aromatic G P P G *Nitric acid G F F F Nitric acid, red and white PPPP fuming Nitropropane (95.5%) F P F F Oleic acid VG F G VG Oxalic acid VG VG VG VG Palmitic acid VG VG VG VG Perchloric acid (60%) VG F G G Perchloroethylene F P P G Phenol VG F G F Phosphoric acid VG G VG VG Potassium hydroxide VG VG VG VG Propyl acetate G F G F i-Propyl alcohol VG VG VG VG n-Propyl alcohol VG VG VG VG Sodium hydroxide VG VG VG VG Styrene (100%) P P P F Sulfuric acid G G G G Tetrahydrofuran P F F F *Toluene F P P F Toluene diisocyanate F G G F *Trichloroethylene F F P G Triethanolamine VG G G VG Tung oil VG P F VG Turpentine G F F VG *Xylene P P P F aPerformance varies with glove thickness and duration of contact. An asterisk indicates limited use. Abbreviations: VG, very good; G, good; F, fair; P, poor (do not use).

properties as nondisposables. Select gloves carefully and always look for some evidence that they will protect against the materials being used. Inspect all gloves before every use for possible holes, tears, or weak areas. Never reuse disposable gloves. Clean reusable gloves after each use and dry carefully inside and out. Observe all common-sense precautions—e.g., do not pipet by mouth, keep unauthorized persons away from hazard- ous chemicals, do not eat or drink in the lab, wear proper clothing in the lab (sandals, open-toed shoes, and shorts are not appropriate). Order hazardous chemicals only in quantities that are likely to be used in a reasonable time. Buying large quantities at a lower unit cost is no bargain if someone (perhaps you) has to pay to dispose of surplus quantities. Substitute alcohol-filled thermometers for mercury-filled thermometers, which are a hazardous chemical spill waiting to happen. Standard Measurements, Data, and Abbreviations A.1H.9

Current Protocols in Molecular Biology Supplement 58 Although any number of chemicals commonly used in laboratories are toxic if used improperly, the toxic properties of a number of reagents require special mention. Chemicals that exhibit carcinogenic, corrosive, flammable, lachrymatory, mutagenic, oxidizing, tera- togenic, toxic, or other hazardous properties are listed in Table A.1H.1. Chemicals listed as carcinogenic range from those accepted by expert review groups as causing cancer in humans to those for which only minimal evidence of carcinogenicity exists. No effort has been made to differentiate the carcinogenic potential of the compounds in Table A.1H.1. Oxidizers may react violently with oxidizable material (e.g., hydrocarbons, wood, and cellulose). Before using any of these chemicals, thoroughly investigate all its charac- teristics. Material Safety Data Sheets are readily available; they list some hazards but vary widely in quality. A number of texts describing hazardous properties are listed at the end of this unit (see Literature Cited). In particular, Sax’s Dangerous Properties of Industrial Materials, 10th ed. (Lewis, 1999) and the Handbook of Reactive Chemical Hazards, 6th ed. (Bretherick et al., 1999) give comprehensive listings of known hazardous properties; however, these texts list only the known properties. Many chemicals, especially fluoro- chromes, have been tested only partially or not at all. Prudence dictates that, unless there is good reason for believing otherwise, all chemicals should be regarded as volatile, highly toxic, flammable human carcinogens and should be handled with great care.

Waste should be segregated according to institutional requirements, for example, into solid, aqueous, nonchlorinated organic, and chlorinated organic material, and should always be disposed of in accordance with all applicable federal, state, and local regula- tions. Extensive information and cautionary details along with techniques for the disposal of chemicals in laboratories have been published (Bretherick, 1986; Lunn and Sansone, 1994a; O’Neil, 2001; Furr, 2000). Some commonly used disposal procedures are outlined in Basic Protocols 1 to 11. Incorporation of these procedures into laboratory protocols can help to minimize waste disposal problems. Alternate Protocols 1 to 7 describe decontamination methods for some of the chemicals. Support Protocols 1 to 9 describe analytical techniques that are used to verify that reagents have been decontaminated; with modification, these assays may also be used to determine the concentration of a particular chemical.

DISPOSAL METHODS A number of procedures for the disposal of hazardous chemicals are available; protocols for the disposal and decontamination of some hazardous chemicals commonly encoun- tered in molecular biology laboratories are listed in Table A.1H.4. These procedures are necessarily brief; for full details consult the original references or a collection of these procedures (see Lunn and Sansone, 1994a). CAUTION: These disposal methods should be carried out only in a chemical fume hood by workers equipped with eye protection, a lab coat, and gloves. Additional protective equipment may be necessary.

BASIC DISPOSAL OF BENZIDINE AND DIAMINOBENZIDINE PROTOCOL 1 Benzidine and diaminobenzidine can be degraded by oxidation with potassium perman- ganate (Castegnaro et al., 1985; Lunn and Sansone, 1991a). This protocol presents a method for decontamination of benzidine and diaminobenzidine in bulk. This method can also be adapted to the decontamination of benzidine and diaminobenzidine spills (see Alternate Protocol 1). These compounds can also be removed from solution using horseradish peroxidase in the presence of hydrogen peroxide (see Alternate Protocol 2). Safe Use of Destruction and decontamination are >99%. Support Protocol 1 is used to test for the Hazardous Chemicals presence of benzidine and diaminobenzidine. A.1H.10

Supplement 58 Current Protocols in Molecular Biology Table A.1H.4 Protocols for Disposal of Some Hazardous Chemicals

Protocol Disposal method for Basic Protocol 1 Benzidine and diaminobenzidine Alternate Protocol 1 Spills of benzidine and diaminobenzidine Alternate Protocol 2 Aqueous solutions of benzidine and diaminobenzidine Support Protocol 1 Analysis for benzidine and diaminobenzidine Basic Protocol 2 Biological stains Alternate Protocol 3 Large volumes of dilute biological stains Support Protocol 2 Analysis for biological stains Basic Protocol 3 Silanes Basic Protocol 4 Cyanide and cyanogen bromide Support Protocol 3 Analysis for cyanide Basic Protocol 5 Dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, 1,3-propane sultone Support Protocol 4 Analysis for dimethyl sulfate, diethyl sulfate, methyl methanesul- fonate, ethyl methanesulfonate, diepoxybutane, 1,3-propane sultone Basic Protocol 6 Ethidium bromide and propidium iodide Alternate Protocol 4 Equipment contaminated with ethidium bromide Alternate Protocol 5 Ethidium bromide in isopropanol containing cesium chloride Alternate Protocol 6 Ethidium bromide in alcohols Support Protocol 5 Analysis for ethidium bromide and propidium iodide Basic Protocol 7 Hydrogen peroxide Basic Protocol 8 Iodine Basic Protocol 9 Mercury compounds Alternate Protocol 7 Waste water containing mercury Support Protocol 6 Analysis for mercury Basic Protocol 10 Sodium azide Support Protocol 7 Analysis for sodium azide Support Protocol 8 Analysis for nitrite Basic Protocol 11 Enzyme inhibitors Support Protocol 9 Analysis for enzyme inhibitors

Materials Benzidine or diaminobenzidine tetrahydrochloride dihydrate 0.1 M HCl (for benzidine) 0.2 M potassium permanganate: prepare immediately before use 2 M sulfuric acid Sodium metabisulfite 10 M potassium hydroxide (KOH) Additional reagents and equipment for testing for the presence of aromatic amines (see Support Protocol 1) 1. For each 9 mg benzidine, add 10 ml of 0.1 M HCl or for each 9 mg diaminobenzidine Standard tetrahydrochloride dihydrate, add 10 ml water. Stir the solution until the aromatic Measurements, amine has completely dissolved. Data, and Abbreviations A.1H.11

Current Protocols in Molecular Biology Supplement 58 2. For each 10 ml of solution, add 5 ml freshly prepared 0.2 M potassium permanganate and 5 ml of 2 M sulfuric acid. Allow the mixture to stand for ≥10 hr. 3. Add sodium metabisulfite until the solution is decolorized. 4. Add 10 M KOH to make the solution strongly basic, pH >12. CAUTION: This reaction is exothermic. 5. Dilute with 5 vol water and pass through filter paper to remove manganese com- pounds. 6. Test the filtrate for the presence of aromatic amines (i.e., benzidine or diaminobenzid- ine; see Support Protocol 1). 7. Neutralize the filtrate with acid and discard.

ALTERNATE DECONTAMINATION OF SPILLS INVOLVING BENZIDINE AND PROTOCOL 1 DIAMINOBENZIDINE Additional Materials (also see Basic Protocol 1) Glacial acetic acid 1:1 (v/v) 0.2 M potassium permanganate/2 M sulfuric acid: prepare immediately before use Absorbent material (e.g., paper towels, Kimwipes) High-efficiency particulate air (HEPA) vacuum (Fisher) Additional reagents and equipment for testing for the presence of aromatic amines (see Support Protocol 1) CAUTION: This procedure may damage painted surfaces and Formica. 1. Remove as much of the spill as possible using absorbent material and high-efficiency particulate air (HEPA) vacuuming. 2. Wet the surface with glacial acetic acid until all the amines are dissolved, then add an excess of freshly prepared 1:1 (v/v) 0.2 M potassium permanganate/2 M sulfuric acid to the spill area. Allow the mixture to stand ≥10 hr. 3. Ventilate the area and decolorize with sodium metabisulfite. 4. Mop up the liquid with paper towels. Squeeze the solution out of the towels and collect in a suitable container. Discard towels as hazardous solid waste. 5. Add 10 M KOH to make the solution strongly basic, pH ≥12. CAUTION: This reaction is exothermic. 6. Dilute with 5 vol water and filter through filter paper to remove manganese com- pounds. 7. Test the filtrate for the presence of aromatic amines (i.e., benzidine or diaminobenzid- ine; see Support Protocol 1). 8. Neutralize the filtrate with acid and discard it. 9. Verify complete decontamination by wiping the surface with a paper towel moistened with water and squeezing the liquid out of the towel. Test the liquid for the presence of benzidine or diaminobenzidine (see Support Protocol 1). Repeat steps 1 to 9 as necessary.

Safe Use of Hazardous Chemicals A.1H.12

Supplement 58 Current Protocols in Molecular Biology DECONTAMINATION OF AQUEOUS SOLUTIONS OF BENZIDINE AND ALTERNATE DIAMINOBENZIDINE PROTOCOL 2 The enzyme horseradish peroxidase catalyzes the oxidation of the amine to a radical which diffuses into solution and polymerizes. The polymers are insoluble and fall out of solution. Additional Materials (also see Basic Protocol 1) Aqueous solution of benzidine or diaminobenzidine 1 N HCl or NaOH 3% (v/v) hydrogen peroxide Horseradish peroxidase (see recipe) 1:1 (v/v) 0.2 M potassium permanganate/2 M sulfuric acid 5% (w/v) ascorbic acid Porous glass filter or Sorvall GLC-1 centrifuge or equivalent Additional reagents and equipment for testing for the presence of aromatic amines (see Support Protocol 1) 1. Adjust the pH of the aqueous benzidine or diaminobenzidine solution to 5 to 7 with 1 N HCl or NaOH as required and dilute so the concentration of aromatic amines is ≤100 mg/liter. 2. For each liter of solution, add 3 ml of 3% hydrogen peroxide and 300 U horseradish peroxidase. Let the mixture stand 3 hr. 3. Remove the precipitate by filtering the solution through a porous glass filter or by centrifuging 5 min at room temperature in a benchtop centrifuge to pellet the precipitate. The precipitate is mutagenic and should be treated as hazardous waste. 4. Immerse the porous glass filter in 1:1 (v/v) 0.2 M potassium permanganate/2 M sulfuric acid. Clean the filter in a conventional fashion and discard potassium permanganate/sulfuric acid solution as described for benzidine and diaminobenzid- ine (see Basic Protocol 1). 5. For each liter of filtrate, add 100 ml of 5% ascorbic acid. 6. Test the filtrate for the presence of aromatic amines (see Support Protocol 1). 7. Discard the decontaminated filtrate.

ANALYTICAL PROCEDURES TO DETECT BENZIDINE AND SUPPORT DIAMINOBENZIDINE PROTOCOL 1 Reversed-phase HPLC (Snyder et al., 1997) is used to test for the presence of aromatic amines. The limit of detection is 1 µg/ml for benzidine and 0.25 µg/ml for diaminobenzid- ine. Materials Decontaminated aromatic amine solution 10:30:20 (v/v/v) acetonitrile/methanol/1.5 mM potassium phosphate buffer (1.5 mM K2HPO4/1.5 mM KH2PO4) (benzidine) or 75:25 (v/v) methanol/1.5 mM potassium phosphate buffer (diaminobenzidine) 250-mm × 4.6-mm-i.d. Microsorb C-8 reversed-phase HPLC column (Varian) or equivalent Standard Measurements, Additional reagents and equipment for reversed-phase liquid chromatography Data, and (Snyder et al., 1997) Abbreviations A.1H.13

Current Protocols in Molecular Biology Supplement 58 Analyze the decontaminated aromatic amine solution by reversed-phase HPLC using a 250-mm × 4.6-mm-i.d. Microsorb C-8 column or equivalent. To detect benzidine, elute with 10:30:20 (v/v/v) acetonitrile/methanol/1.5 mM potassium phosphate buffer at a flow rate of 1.5 ml/min and UV detection at 285 nm. To detect diaminobenzidine, elute with 75:25 (v/v) methanol/1.5 mM potassium phosphate buffer at a flow rate of 1 ml/min and UV detection at 300 nm.

BASIC DISPOSAL OF BIOLOGICAL STAINS PROTOCOL 2 Biological stains (Table A.1H.5), as well as ethidium bromide and propidium iodide, can be removed from solution using the polymeric resin Amberlite XAD-16. The decontami- nated solution may be disposed of as nonhazardous aqueous waste and the resin as hazardous solid waste. The volume of contaminated resin generated is much smaller than the original volume of the solution of biological stain, so the waste disposal problem is greatly reduced. The final concentration of any remaining stain should be less than the limit of detection (see Support Protocol 2 and Table A.1H.5). In each case decontamina- tion should be >99%. This protocol describes a method for batch decontamination in which the resin is stirred in the solution to be decontaminated and removed by filtration at the end of the reaction time. Large volumes of biological stain can be decontaminated using a column (see Alternate Protocol 3). For full details refer to the original literature (Lunn and Sansone, 1991b) or a compilation (Lunn and Sansone, 1994a).

Table A.1H.5 Decontamination of Biological Stains

Time required for Volume of solution Compound complete (ml) decontaminated decontamination per gram resin Acridine orange 18 hr 20 Alcian blue 8GX 10 min 500 Alizarin red S18 hr 5 Azure A 10 min 80 Azure B 10 min 80 Brilliant blue R 2 hr 80 Congo red 2 hr 40 Coomassie brilliant blue G 2 hr 80 Cresyl violet acetate 2 hr 40 Crystal violet 30 min 200 Eosin B 30 min 40 Erythrosin B 18 hr 10 Ethidium bromide 4 hr 20 Janus green B 30 min 80 Methylene blue 30 min 80 Neutral red 10 min 500 Nigrosin 2 hr 80 Orcein 2 hr 200 Propidium iodide 2 hr 20 Rose Bengal 3 hr 20 Safranine O 1 hr 20 Toluidine blue O 30 min 80 Safe Use of Trypan blue 2 hr 40 Hazardous Chemicals A.1H.14

Supplement 58 Current Protocols in Molecular Biology Materials Amberlite XAD-16 resin (Supelco) 100 µg/ml biological stain in water Additional reagents and equipment for testing for the presence of biological stain (see Support Protocol 2)

For batch decontamination of 20 ml stain 1a. Add 1 g Amberlite XAD-16 to 20 ml of 100 µg/ml biological stain in water. For aqueous solutions having stain concentrations other than 100 ìg/ml, use proportion- ately greater or lesser amounts of resin to achieve complete decontamination. For solutions of erythrosin B, use 2 g Amberlite XAD-16 for 20 ml stain. 2a. Stir the mixture for at least the time indicated in Table A.1H.5.

For batch decontamination of larger volumes of stain 1b. Add 1 g Amberlite XAD-16 to the volume of a 100 µg/ml biological stain in water indicated in Table A.1H.5. 2b. Stir the mixture for at least 18 hr. 3. Remove the resin by filtration through filter paper. 4. Test the filtrate for the presence of the biological stain (see Support Protocol 2). 5. Discard the resin as hazardous solid waste and the decontaminated filtrate as liquid waste.

CONTINUOUS-FLOW DECONTAMINATION OF AQUEOUS ALTERNATE SOLUTIONS OF BIOLOGICAL STAINS PROTOCOL 3 For treating large volumes of dilute aqueous solutions of biological stains (Table A.1H.5), it is possible to put the resin in a column and run the contaminated solution through the column in a continuous-flow system (Lunn et al., 1994). Limited grinding of the Amberlite XAD-16 resin increases its efficiency. Additional Materials (also see Basic Protocol 2) 25 µg/ml biological stain in water Methanol (optional) 300-mm × 11-mm-i.d. glass chromatography column fitted with threaded adapters and flow-regulating valves at top and bottom nut and insert connectors, and insertion tool (Ace Glass) or 300-mm × 15-mm-i.d. glass chromatography column (Spectrum 124010, Fisher) Glass wool 1.5-mm-i.d. × 0.3-mm-wall Teflon tubing Waring blender (optional) Rubber stopper fitted over a pencil QG 20 lab pump (Fluid Metering) Additional reagents and equipment for testing for the presence of biological stain (see Support Protocol 2)

Using a slurry of Amberlite XAD-16 1a. Prepare a 300-mm × 11-mm-i.d. glass chromatography column. To prevent clogging of the column outlet, place a small plug of glass wool at the bottom of the chroma- Standard × Measurements, tography column. Connect 1.5-mm-i.d. 0.3-mm wall Teflon tubing to the adapters Data, and using nut and insert connectors. Attach the tubing using an insertion tool. Abbreviations A.1H.15

Current Protocols in Molecular Biology Supplement 58 Table A.1H.6 Breakthrough Volumes for Continuous-Flow Decontamination of Biological Stains

Breakthrough volume (ml) Compound Limit of 1 ppm 5 ppm detection Acridine orange 465 >990 >990 Alizarin red S120 150 240 Azure A 615 810 >975 Azure B 630 882 >1209 Cresyl violet acetate 706 >1396 >1396 Crystal violet 1020 >1630 >1630 Ethidium bromide 260 312 416 Janus green B 170 650 >870 Methylene blue 420 645 1050 Neutral red >2480 >2480 >2480 Safranine O 365 438 584 Toluidine blue O 353 494 606

2a. Mix 10 g Amberlite XAD-16 and 25 ml water in a beaker and stir 5 min to wet the resin.

Using a finely ground Amberlite XAD-16 slurry 1b. Prepare a 300-mm × 15-mm-i.d. glass chromatography column. To prevent clogging of the column outlet, place a small plug of glass wool at the bottom of the chroma- tography column. 2b. Grind 20 g Amberlite XAD-16 with 200 ml water for exactly 10 sec in a Waring blender. 3. Pour the resin slurry into the column through a funnel. As the resin settles, tap the column with a rubber stopper fitted over a pencil to encourage even packing. Attach a QG 20 lab pump. 4. Pump the 25-µg/ml biological stain solution through the column at 2 ml/min. Alternatively, gravity flow coupled with periodic adjustment of the flow-regulating valve can be used. 5. Check the effluent from the column for the presence of biological stain (see Support Protocol 2). Stop the pump when stain is detected. Table A.1H.6 lists breakthrough volumes at different detection levels for a number of biological stains. 6. Discard the decontaminated effluent and the contaminated resin appropriately. 7. Many biological stains can be washed off the resin with methanol so the resin can be reused. Discard the methanol solution of stain as hazardous organic liquid waste.

Safe Use of Hazardous Chemicals A.1H.16

Supplement 58 Current Protocols in Molecular Biology Table A.1H.7 Methods for Detecting Biological Stainsa

Limit of Compound Reagentb Procedure Wavelength(s)(nm) detection (ppm) Acridine orange DNA solution F ex 492, em 528 0.0032 Alcian blue 8GX A 615 0.9 Alizarin red S1 M KOH A 556 0.46 Azure A A 633 0.15 Azure B A 648 0.13 Brilliant blue R A 585 1.0 Congo red A 497 0.25 Coomassie brilliant A 610 1.7 blue G Cresyl violet acetate pH 5 buffer F ex 588, em 618 0.021 Crystal violet A 588 0.1 Eosin B A 514 0.21 Erythrosin B F ex 488, em 556 0.025 Ethidium bromide F ex 540, em 590 0.05 Janus green B A 660 0.6 Methylene blue A 661 0.13 Neutral red pH 5 buffer A 540 0.6 Nigrosin A 570 0.8 Orcein 1 M KOH A 579 1.15 Propidium iodide DNA solution F ex 350, em 600 0.1 Rose Bengal F ex 520, em 576 0.04 Safranine O F ex 460, em 582 0.03 Toluidine blue O A 626 0.2 Trypan blue A 607 0.22 aAbbreviations: A, absorbance; em, emission; ex, excitation; F, fluorescence bSee Support Protocol 2

ANALYTICAL PROCEDURES TO DETECT BIOLOGICAL STAIN SUPPORT PROTOCOL 2 Depending on the biological stain, the filtrate or eluate from the decontamination procedure can be analyzed using either UV absorption (A) or fluorescence detection (F). Materials Filtrate or eluate from biological stain decontamination (see Basic Protocol 2 or Alternate Protocol 3) pH 5 buffer (see recipe) 1 M KOH solution 20 µg/ml calf thymus DNA in TBE electrophoresis buffer, pH 8.1 (APPENDIX 2A) Test the filtrate or eluate from the biological stain decontamination procedure using the appropriate method as indicated in Table A.1H.7.

Traces of acid or base on the resin may induce color changes in the stain. Accordingly, with cresyl violet acetate or neutral red, mix aliquots of the filtrate with 1 vol pH 5 buffer before analyzing. With alizarin red S and orcein, mix aliquots of the filtrate with 1 vol of 1 M KOH solution before analyzing. Increase the fluorescence of solutions of acridine orange, ethidium bromide, and propidium ì Standard iodide by mixing an aliquot of the filtrate with an equal volume of 20 g/ml calf thymus Measurements, DNA in TBE electrophoresis buffer, pH 8.1. Let the solution stand 15 min before measuring Data, and the fluorescence. Abbreviations A.1H.17

Current Protocols in Molecular Biology Supplement 58 BASIC DISPOSAL OF CHLOROTRIMETHYLSILANE AND PROTOCOL 3 DICHLORODIMETHYLSILANE Silane-containing compounds are hydrolyzed to hydrochloric acid and polymeric silicon- containing material (Patnode and Wilcock, 1946). 1. Hydrolyze silane-containing compounds by cautiously adding 5 ml silane to 100 ml vigorously stirred water in a flask. Allow the resulting suspension to settle. 2. Remove any insoluble material by filtration and discard it with the solid or liquid hazardous waste. 3. Neutralize the aqueous layer with base and discard it.

BASIC DISPOSAL OF CYANIDES AND CYANOGEN BROMIDE PROTOCOL 4 Inorganic cyanides (e.g., NaCN) and cyanogen bromide (CNBr) are oxidized by sodium hypochlorite (NaOCl; e.g., Clorox) in basic solution to the much less toxic cyanate ion (Lunn and Sansone, 1985a). Destruction is >99.7%. Materials Cyanide (e.g., NaCN) or cyanogen bromide (CNBr) 1 M NaOH 5.25% (v/v) sodium hypochlorite (NaOCl; i.e., standard household bleach) Additional reagents and equipment for testing for the presence of cyanide (see Support Protocol 3) 1. Dissolve cyanide in water to give a concentration ≤25 mg/ml or dissolve CNBr in water to give a concentration ≤60 mg/ml. If necessary, dilute aqueous solutions so the concentration of NaCN or CNBr does not exceed the limit. 2. Mix 1 vol NaCN or CNBr solution with 1 vol 1 M NaOH and 2 vol fresh 5.25% NaOCl. Stir the mixture 3 hr. IMPORTANT NOTE: With age bleach may become ineffective; use of fresh bleach is strongly recommended. 3. Test the reaction mixture for the presence of cyanide (see Support Protocol 3). 4. Neutralize the reaction mixture and discard it.

SUPPORT ANALYTICAL PROCEDURE TO DETECT CYANIDE PROTOCOL 3 This protocol is used to detect cyanide or cyanogen bromide at ≥3 µg/ml. Materials Cyanide or cyanogen bromide decontamination reaction mixture (see Basic Protocol 4) Phosphate buffer (see recipe) 10 mg/ml sodium ascorbate in water: prepare fresh daily 100 mg/liter NaCN in water: prepare fresh weekly 10 mg/ml chloramine-T in water: prepare fresh daily Cyanide detection reagent (see recipe) Sorvall GLC-1 centrifuge or equivalent

Safe Use of Hazardous Chemicals A.1H.18

Supplement 58 Current Protocols in Molecular Biology 1. If necessary to remove suspended solids, centrifuge two 1-ml aliquots of the cyanide or cyanogen bromide decontamination reaction mixture 5 min in a benchtop centri- fuge, room temperature. Add each supernatant to 4 ml phosphate buffer in separate tubes. 2. If an orange or yellow color appears, add 10 mg/ml freshly prepared sodium ascorbate dropwise until the mixture is colorless, but do not add more than 2 ml. 3. Add 200 µl of 100 mg/liter NaCN to one reaction mixture (control solution). 4. Add 1 ml freshly prepared 10 mg/ml chloramine-T to each tube. Shake the tubes and let them stand 1 to 2 min. 5. Add 1 ml cyanide detection reagent, shake, and let stand 5 min. A blue color indicates the presence of cyanide. If destruction has been complete and the analytical procedure has been carried out correctly, the treated reaction mixture should be colorless and the control solution, which contains NaCN, should be blue. 6. Centrifuge tubes 5 min, room temperature, if necessary to remove suspended solids. Measure the absorbance at 605 nm with appropriate standards and blanks.

DISPOSAL OF DIMETHYL SULFATE, DIETHYL SULFATE, METHYL BASIC METHANESULFONATE, ETHYL METHANESULFONATE, PROTOCOL 5 DIEPOXYBUTANE, AND 1,3-PROPANE SULTONE Dimethyl sulfate is hydrolyzed by base to methanol and methyl hydrogen sulfate (Lunn and Sansone, 1985b). Subsequent hydrolysis of methyl hydrogen sulfate to methanol and sulfuric acid is slow. Methyl hydrogen sulfate is nonmutagenic and a very poor alkylating agent. The other compounds can be hydrolyzed with base in a similar fashion (Lunn and Sansone, 1990a). Destruction is >99%. A method to verify that decontamination is complete is also provided (see Support Protocol 4). NOTE: The reaction times given in the protocol should give good results; however, reaction time may be affected by such factors as the size and shape of the flask and the rate of stirring. The presence of two phases indicates that the reaction is not complete, and stirring should be continued until the reaction mixture is homogeneous. Materials Dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, or 1,3-propane sultone 5 M NaOH Additional reagents and equipment for testing for the presence of dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, or 1,3-propane sultone (see Support Protocol 4)

For bulk quantities of dimethyl sulfate 1a. Add 100 ml dimethyl sulfate to 1 liter of 5 M NaOH. Stir the reaction mixture. 2a. Fifteen minutes after all the dimethyl sulfate has gone into solution, neutralize the reaction mixture with acid.

For bulk quantities of diethyl sulfate 1b. Add 100 ml diethyl sulfate to 1 liter of 5 M NaOH. Stir the reaction mixture 24 hr. Standard Measurements, 2b. Neutralize the reaction mixture with acid. Data, and Abbreviations A.1H.19

Current Protocols in Molecular Biology Supplement 58 For bulk quantities of methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, and 1,3-propane sultone 1c. Add 1 ml methyl methanesulfonate, ethyl methanesulfonate, or diepoxybutane, or 1 g of 1,3-propane sultone to 10 ml of 5 M NaOH. Stir the reaction mixture 1 hr for 1,3-propane sultone, 2 hr for methyl methanesulfonate, 22 hr for diepoxybutane, or 24 hr for ethyl methanesulfonate. 2c. Neutralize the reaction mixture with acid. 3. Test the reaction mixture for the presence of the original compound (see Support Protocol 4). 4. Discard the decontaminated reaction mix.

SUPPORT ANALYTICAL PROCEDURE TO DETECT THE PRESENCE OF DIMETHYL PROTOCOL 4 SULFATE, DIETHYL SULFATE, METHYL METHANESULFONATE, ETHYL METHANESULFONATE, DIEPOXYBUTANE, AND 1,3-PROPANE SULTONE This protocol is used to verify decontamination of solutions containing dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, or 1,3-propane sultone. The detection limit for this assay is 40 µg/ml for dimethyl sulfate, 108 µg/ml for diethyl sulfate, 84 µg/ml for methyl methanesulfonate, 1.1 µg/ml for ethyl methanesulfonate, 360 µg/ml for diepoxybutane, and 264 µg/ml for 1,3-propane sultone. Materials Reaction mixture containing dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, or 1,3-propane sultone 98:2 (v/v) 2-methoxyethanol/acetic acid 5% (w/v) 4-(4-nitrobenzyl)pyridine in 2-methoxyethanol Piperidine 2-Methoxyethanol 1. Dilute an aliquot of the reaction mixture with 4 vol water. 2. Add 100 µl diluted reaction mixture to 1 ml of 98:2 (v/v) 2-methoxyethanol/acetic acid. Swirl to mix. 3. Add 1 ml of 5% (w/v) 4-(4-nitrobenzyl)pyridine in 2-methoxyethanol. Heat 10 min at 100°C, then cool 5 min in ice. 4. Add 0.5 ml piperidine and 2 ml of 2-methoxyethanol. 5. Measure the absorbance of the violet reaction mixture at 560 nm against an appro- priate blank. The absorbance of a decontaminated solution should be 0.000.

BASIC DISPOSAL OF ETHIDIUM BROMIDE AND PROPIDIUM IODIDE PROTOCOL 6 Ethidium bromide and propidium iodide in water and buffer solutions may be degraded by reaction with sodium nitrite and hypophosphorous acid in aqueous solution (Lunn and Sansone, 1987); destruction is >99.87%. This reaction may also be used to decontaminate equipment contaminated with ethidium bromide (see Alternate Protocol 4; Lunn and Sansone, 1989) and to degrade ethidium bromide in organic solvents (see Alternate Protocol 5 and Alternate Protocol 6; Lunn and Sansone, 1990b). Ethidium bromide and Safe Use of propidium iodide may also be removed from solution by adsorption onto Amberlite Hazardous XAD-16 resin (see Basic Protocol 2). Chemicals A.1H.20

Supplement 58 Current Protocols in Molecular Biology Materials Ethidium bromide– or propidium iodide–containing solution in water, buffer, or 1 g/ml cesium chloride 5% (v/v) hypophosphorous acid: prepare fresh daily by diluting commercial 50% reagent 1/10 0.5 M sodium nitrite: prepare fresh daily Sodium bicarbonate Additional reagents and equipment for testing for the presence of ethidium bromide or propidium iodide (see Support Protocol 5) 1. If necessary, dilute the ethidium bromide– or propidium iodide–containing solution so the concentration of ethidium bromide or propidium iodide is ≤0.5 mg/ml. 2. For each 100 ml solution, add 20 ml of 5% hypophosphorous acid solution and 12 ml of 0.5 M sodium nitrite. Stir briefly and let stand 20 hr. 3. Neutralize the reaction mixture by adding sodium bicarbonate until the evolution of gas ceases. 4. Test the reaction mixture for the presence of ethidium bromide or propidium iodide (see Support Protocol 5). 5. Discard the decontaminated reaction mixture.

DECONTAMINATION OF EQUIPMENT CONTAMINATED WITH ALTERNATE ETHIDIUM BROMIDE PROTOCOL 4 Glass, stainless steel, Formica, floor tile, and the filters of transilluminators have been successfully decontaminated using this protocol. No change in the optical properties of the transilluminator filter could be detected, even after a number of decontamination cycles. Materials Equipment contaminated with ethidium bromide Decontamination solution (see recipe) Sodium bicarbonate Additional reagents and equipment for testing for the presence of ethidium bromide (see Support Protocol 5) 1. Wash the equipment contaminated with ethidium bromide once with a paper towel soaked in decontamination solution. The pH of the decontamination solution is 1.8. If this would be too corrosive for the surface to be decontaminated, wash with a paper towel soaked in water instead. 2. Wash the surface five times with paper towels soaked in water using a fresh towel each time. 3. Soak all the towels 1 hr in decontamination solution. 4. Neutralize the decontamination solution by adding sodium bicarbonate until the evolution of gas ceases. 5. Test the decontamination solution for the presence of ethidium bromide (see Support Protocol 5). Standard Measurements, 6. Discard the decontamination solution and the paper towels as nonhazardous liquid Data, and and solid wastes. Abbreviations A.1H.21

Current Protocols in Molecular Biology Supplement 58 ALTERNATE DECONTAMINATION OF ETHIDIUM BROMIDE IN ISOPROPANOL PROTOCOL 5 SATURATED WITH CESIUM CHLORIDE Materials Ethidium bromide in isopropanol saturated with cesium chloride Decontamination solution (see recipe) Sodium bicarbonate Additional reagents and equipment for testing for the presence of ethidium bromide (see Support Protocol 5) 1. If necessary, dilute the ethidium bromide in isopropanol saturated with cesium chloride so the concentration of ethidium bromide is ≤1 mg/ml. 2. For each volume of ethidium bromide solution, add 4 vol decontamination solution. Stir the reaction mixture 20 hr. 3. Neutralize the reaction mixture by adding sodium bicarbonate until the evolution of gas ceases. 4. Test the reaction mixture for the presence of ethidium bromide (see Support Protocol 5). 5. Discard the decontaminated solution.

ALTERNATE DECONTAMINATION OF ETHIDIUM BROMIDE IN ISOAMYL PROTOCOL 6 ALCOHOL AND 1-BUTANOL Materials Ethidium bromide in isoamyl alcohol or 1-butanol Decontamination solution (see recipe) Activated charcoal Sodium bicarbonate Separatory funnel Additional reagents and equipment for testing for the presence of ethidium bromide 1. If necessary, dilute the ethidium bromide in isoamyl alcohol or 1-butanol so the concentration is ≤1 mg/ml final. 2. For each volume of ethidium bromide solution, add 4 vol decontamination solution. Stir the two-phase reaction mixture rapidly for 72 hr. 3. For each 100 ml of reaction mixture, add 2 g activated charcoal. Stir another 30 min. 4. Filter the reaction mixture. 5. Neutralize the filtrate by adding sodium bicarbonate until the evolution of gas ceases. Separate the layers using a separatory funnel. More alcohol may tend to separate from the aqueous layer on standing. 6. Test the alcohol and aqueous layers for the presence of ethidium bromide. 7. Discard the alcohol and aqueous layers appropriately. Discard the activated charcoal as solid waste. The aqueous layer contains 4.6% 1-butanol or 2.3% isoamyl alcohol.

Safe Use of Hazardous Chemicals A.1H.22

Supplement 58 Current Protocols in Molecular Biology ANALYTICAL PROCEDURE TO DETECT ETHIDIUM BROMIDE OR SUPPORT PROPIDIUM IODIDE PROTOCOL 5 This protocol is used to verify that solutions no longer contain ethidium bromide or propidium iodide. The limits of detection are 0.05 parts per million (ppm) for ethidium bromide and 0.1 ppm for propidium iodide. Materials Reaction mixture containing ethidium bromide or propidium iodide TBE buffer, pH 8.1 (APPENDIX 2A) 20 µg/ml calf thymus DNA in TBE buffer, pH 8.1 1. Mix 100 µl reaction mixture containing ethidium bromide or propidium iodide with 900 µl TBE buffer, pH 8.1. 2. Add 1 ml of 20 µg/ml calf thymus DNA in TBE, pH 8.1. Prepare a blank solution (100 µl water + 900 µl TBE + 1 ml of 20 µg/ml calf thymus DNA) and control solutions containing known quantities of ethidium bromide or propidium iodide. Let the mixtures stand 15 min. 3. To detect ethidium bromide, measure the fluorescence with an excitation wavelength of 540 nm and an emission wavelength of 590 nm. To detect propidium iodide, measure the fluorescence with an excitation wavelength of 350 nm and an emission wavelength of 600 nm. If a spectrophotofluorometer is not available, fluorescence of ethidium bromide can be qualitatively determined using a hand-held UV lamp on the long-wavelength setting (Lunn and Sansone, 1991c).

DISPOSAL OF HYDROGEN PEROXIDE BASIC Hydrogen peroxide can be reduced with sodium metabisulfite (Lunn and Sansone, PROTOCOL 7 1994b). Materials 30% hydrogen peroxide 10% (w/v) sodium metabisulfite 10% (w/v) potassium iodide 1 M HCl 1% (w/v) starch indicator solution 1. Add 5 ml of 30% hydrogen peroxide to 100 ml of 10% sodium metabisulfite. Stir the mixture at room temperature until the temperature starts to drop, indicating that the reaction is over. 2. Test for the presence of hydrogen peroxide by adding a few drops of the reaction mixture to an equal volume of 10% potassium iodide. Add a few drops of 1 M HCl to acidify the reaction mixture, then add a drop of 1% starch indicator solution. A deep blue color indicates the presence of excess oxidant. If necessary, add more 10% sodium metabisulfite until the starch test is negative. 3. Discard the decontaminated mixture.

Standard Measurements, Data, and Abbreviations A.1H.23

Current Protocols in Molecular Biology Supplement 58 BASIC REDUCTION OF IODINE PROTOCOL 8 Iodine is reduced with sodium metabisulfite to iodide (Lunn and Sansone, 1994b). Materials Iodine 10% (w/v) sodium metabisulfite 1 M HCl 1% (w/v) starch indicator solution 1. Add 5 g iodine to 100 ml of 10% sodium metabisulfite. Stir the mixture until the iodine has completely dissolved. 2. Acidify a few drops of the reaction mixture by adding a few drops of 1 M HCl. Add 1 drop of 1% starch indicator solution. A deep blue color indicates the presence of iodine. If reduction is not complete, add more sodium metabisulfite solution. 3. Dispose of the decontaminated solution.

BASIC DISPOSAL OF MERCURY COMPOUNDS PROTOCOL 9 Solutions of mercuric acetate can be decontaminated using Dowex 50X8-100, a strongly acidic gel-type ion-exchange resin with a sulfonic acid functionality. Solutions of mercu- ric chloride can be decontaminated using Amberlite IRA-400(Cl), a strongly basic gel-type ion-exchange resin with a quaternary ammonium functionality. The final con- centration of mercury is <3.8 ppm (Lunn and Sansone, 1994a). On a small scale it is most convenient to stir the resin in the solution to be decontaminated, but on a larger scale, or for routine use, it may be more convenient to pass the solution through a column packed with the resin. Although the volume of waste that must be disposed of is greatly reduced using this technique, a small amount of waste (i.e., the resin contaminated with mercury) remains and must be discarded appropriately. Resin can be regenerated by washing with acid, but the concentrated metal-containing solution generated by this must also be disposed of appropriately. Mercury may also be removed from laboratory waste water using a column of iron powder (see Alternate Protocol 7). Support Protocol 6 is used to detect the presence of mercury. Materials Solution containing ≤1600 ppm mercuric acetate or ≤1350 ppm mercuric chloride Dowex 50X8-100 ion-exchange resin or Amberlite IRA-400(Cl) ion-exchange resin Additional reagents and equipment to test for the presence of mercury (see Support Protocol 6) 1a. For mercuric acetate: For each 200 ml of solution containing ≤1600 ppm mercuric acetate, add 1 g Dowex 50X8-100 ion-exchange resin. Stir the mixture 1 hr, then filter through filter paper. 1b. For mercuric chloride: For each 200 ml of solution containing ≤1350 ppm mercuric chloride, add 1 g Amberlite IRA-400(Cl) ion-exchange resin. Stir the mixture 6 hr, then filter through filter paper. The speed and efficiency of decontamination will depend on factors such as the size and shape of the flask and the rate of stirring.

Safe Use of 3. Test the filtrate for the presence of mercury (see Support Protocol 6). Hazardous Chemicals 4. Discard the decontaminated filtrate and the mercury-containing resin appropriately. A.1H.24

Supplement 58 Current Protocols in Molecular Biology DECONTAMINATION OF WASTE WATER CONTAINING MERCURY ALTERNATE PROTOCOL 7 Laboratory waste water that contains mercury is decontaminated by passing it through a column of iron powder. The mercury forms mercury amalgam and stays on the column. Some metallic mercury remains in solution but this can be removed by aeration. The final concentration of mercury is <5 ppb (Shirakashi et al., 1986). Materials Iron powder, 60 mesh Waste water containing ≤2.5 ppm mercury 6-mm-i.d. column 1. Pack a 6-mm-i.d. column with 1 g of 60-mesh iron powder. Use a fresh column for each treatment. 2. Pass ≤2 liters of water containing ≤2.5 ppm of mercury through the column at a flow rate of 250 ml/hr. Solutions containing a higher concentration of mercury may also be treated, but this will result in a higher final concentration of mercury (e.g., treating a 100-ppm solution in this fashion yielded 33 ppb final). Some iron ends up in solution and can be removed by adjusting the pH to 8. The resulting precipitated Fe(OH)3 can then be removed by filtration. 3. Aerate the resulting effluent to remove traces of metallic mercury and continue aeration 30 min after the last of the effluent has emerged from the column. Vent the metallic mercury removed from the solution by aeration into the fume hood or capture it in a mercury trap. The effluent contains <5 ppb mercury. The presence of iodide or polypeptone may neces- sitate several treatments to reduce the mercury to an acceptable level.

ANALYTICAL PROCEDURE TO DETECT MERCURY SUPPORT Atomic absorption spectroscopy with detection at 253.7 nm, a slit width of 0.7 nm, and PROTOCOL 6 a limit of detection of 3.8 ppm can be used to determine the concentration of mercury in solution for experiments involving ion-exchange resins. A Hiranuma mercury meter model HG-1 can be used for experiments involving iron powder.

DISPOSAL OF SODIUM AZIDE BASIC Sodium azide can be oxidized by ceric ammonium nitrate (Manufacturing Chemists PROTOCOL 10 Association, 1973) to nitrogen (Mason, 1967) or by nitrous acid (National Research Council, 1983) to nitrous oxide (Mason, 1967); destruction is >99.996%. Sodium azide in buffer solution may also be degraded by the addition of sodium nitrite (Lunn and Sansone, 1994a). The reaction proceeds much more readily at low pH, but if sufficient sodium nitrite is added, it will proceed to completion even at high pH. At low pH, it may be possible to completely degrade the azide present in the buffer with less than the amount of sodium nitrite indicated. However, the reaction mixture must be carefully checked to ensure that no azide remains (see Support Protocol 7). At high pH it is possible for unreacted azide to remain in the presence of excess nitrite. Residual nitrite can be detected using Support Protocol 8. CAUTION: Some toxic nitrogen dioxide may be produced as a by-product of these reactions, so they should always be carried out in a chemical fume hood. Standard Measurements, Data, and Abbreviations A.1H.25

Current Protocols in Molecular Biology Supplement 58 Materials Sodium azide or solution containing sodium azide Ceric ammonium nitrate 10% (w/v) potassium iodide 1 M HCl 1% (w/v) starch indicator solution Sodium nitrite 4 M sulfuric acid Additional reagents and equipment to test for the presence of sodium azide (see Support Protocol 7) or nitrite (see Support Protocol 8)

Decontamination using ceric ammonium nitrate 1a. For each gram of sodium azide, add 9 g ceric ammonium nitrate to 30 ml of water, and stir until it has dissolved. 2a. Dissolve each gram of sodium azide in 5 ml water. Add this solution to the ceric ammonium nitrate solution at the rate of 1 ml each min. Stir 1 hr. If the reaction is carried out on a larger scale, an ice bath may be required for cooling. 3a. Check that the reaction is still oxidizing by adding a few drops of the reaction mixture to an equal volume of 10% potassium iodide. Acidify the mixture with 1 drop 1 M HCl and add 1 drop 1% starch indicator solution. The deep blue color of the starch-iodine complex indicates that excess oxidant is present. If excess oxidant is not present, add more ceric ammonium nitrate. 4a. Test for the presence of sodium azide (see Support Protocol 8). 5a. Discard the decontaminated reaction mixture.

Decontamination using sodium nitrite 1b. For each 5 g sodium azide, dissolve 7.5 g sodium nitrite in 30 ml water. 2b. Dissolve each 5 g sodium azide in 100 ml water. Add the sodium nitrite solution with stirring. Slowly add 4 M sulfuric acid until the reaction mixture is acidic to litmus. Stir 1 hr. CAUTION: It is important to add the sodium nitrite, then the sulfuric acid. Adding these reagents in reverse order will generate explosive, volatile, toxic hydrazoic acid. If the reaction is carried out on a large scale, an ice bath may be required for cooling. 3b. Check that there is excess nitrous acid in the reaction. Add a few drops of the reaction mixture to an equal volume of 10% potassium iodide. Acidify the mixture with 1 drop 1 M HCl. Add 1 drop starch indicator solution. The deep blue color of the starch-iodine complex indicates that excess nitrous acid is present. If excess nitrous acid is not present, add more sodium nitrite. 4b. If excess nitrous acid is present, test for the presence of sodium azide (see Support Protocol 7). 5b. Discard the decontaminated reaction mixture.

Decontamination of sodium azide in buffer 1c. If necessary, dilute the buffer solution with water so the concentration of sodium azide Safe Use of is ≤1 mg/ml. Hazardous Chemicals 2c. For each 50 ml buffer solution add 5 g sodium nitrite. Stir the reaction 18 hr. A.1H.26

Supplement 58 Current Protocols in Molecular Biology 3c. Test for the presence of sodium azide (see Support Protocol 7). 4c. Discard the decontaminated reaction solution.

ANALYTICAL PROCEDURES TO DETECT SODIUM AZIDE SUPPORT PROTOCOL 7 Sodium azide is analyzed by reacting azide ion with 3,5-dinitrobenzoyl chloride to form 3,5-dinitrobenzoyl azide, which can be detected by reversed-phase HPLC. The limit of detection of this assay is 0.2 µg/ml sodium azide. This protocol works only in the absence of nitrite; verify that all of the nitrite has been destroyed by sulfamic acid by using the method detailed later in this unit (see Support Protocol 8). Materials Reaction mixture from sodium azide treated with ceric ammonium nitrate or sodium nitrite 1 M KOH Acetonitrile Sodium azide indicator solution (see recipe) 0.2 M HCl 20% (w/v) sulfamic acid 3,5-dinitrobenzoyl chloride 50:50 (v/v) acetonitrile/water Sorvall GLC-1 centrifuge or equivalent 25-cm × 4.6-mm-i.d. Microsorb C-8 reversed-phase HPLC column (Varian) or equivalent Additional reagents and equipment for reversed-phase liquid chromatography (Snyder et al., 1997)

To analyze for azide in the presence of ceric salts 1a. To a 10-ml aliquot of the reaction mixture from sodium azide treated with ceric ammonium nitrate add 40 ml water. Add 5 ml of this diluted solution to 3 ml of 1 M KOH and mix by shaking. If <3 ml of 1 M KOH is used, precipitation of ceric salts will not be complete. 2a. Centrifuge the mixture 5 min, room temperature. 3a. Remove 2 ml supernatant and add to 1 ml acetonitrile. Add 1 drop sodium azide indicator solution, add 0.2 M HCl dropwise until the mixture turns yellow, then add 1 drop more. To analyze for azide in the presence of nitrite 1b. To 5 ml of the reaction mixture from sodium azide treated with sodium nitrite add ≥1 ml sulfamic acid to remove excess nitrite. Let stand ≥3 min. More sulfamic acid solution may be required for strongly basic reaction mixtures or those containing high concentrations of nitrite. Complete removal of nitrite can be checked by using a modified Griess reagent (see Support Protocol 8). At high pH the reaction between azide and nitrite is quite slow, so the presence of excess nitrite does not mean that all the azide has been degraded. 2b. Add 1 drop sodium azide indicator solution, then basify the mixture by adding 1 M KOH until it turns purple (typically, 3 to 10 ml are required). 3b. Add 2 ml acetonitrile. Add 0.2 M HCl dropwise until the mixture turns yellow, then Standard add 1 drop more. Measurements, Data, and If >1 ml sulfamic acid is used, add 4 ml acetonitrile. Abbreviations A.1H.27

Current Protocols in Molecular Biology Supplement 58 4. Prepare a 10% (w/v) solution of 3,5-dinitrobenzoyl chloride in acetonitrile. 5. Add 50 µl of 10% dinitrobenzoyl chloride/acetonitrile to the reaction mix (step 3a or 3b). Shake the mixture and let it stand ≥3 min. Longer standing times have no effect on the HPLC analysis. However, it is crucial to use freshly prepared 3,5-dinitrobenzoyl chloride solution within minutes of its preparation. It is generally most convenient to prepare all the analytical samples with the fresh solution at the beginning of the day and analyze them over the course of the day. 6. Analyze 20 µl of each reaction mixture by reversed-phase HPLC (Snyder et al., 1997) using a mobile phase of 50:50 (v/v) acetonitrile/water with a flow rate of 1 ml/min and UV detection at 254 nm. The peak for 3,5-dinitrobenzoyl azide elutes at ∼9 min.

SUPPORT ANALYTICAL PROCEDURE TO DETECT NITRITE PROTOCOL 8 This protocol uses a modified Griess reagent to test for the presence of nitrite. The limit of detection of this assay is 0.06 µg/ml nitrite. A similar procedure uses N-(1-naphthyl)- ethylenediamine (Cunniff, 1995). Materials α-Naphthylamine 15% (v/v) aqueous acetic acid Sulfanilic acid solution (see recipe) Reaction mixture treated to remove excess nitrite (see Support Protocol 7, step 1b)

Table A.1H.8 Conditions for the Destruction of Enzyme Inhibitors

Solution: Compound Concentration Solvent Time 1 M NaOH AEBSF 1 mM Buffer(pH 5.0-8.0) 1:0.1 1 hr AEBSF 20 mM DMSO 1:10 24 hr AEBSF 20 mM Isopropanol 1:10 24 hr APMSF 2.5 mM Buffer(pH 5.0-8.0) 1:0.1 1 hr APMSF 25 mM DMSO 1:5 24 hr APMSF 25 mM 50:50 isopropanol:pH 3 buffer 1:5 24 hr APMSF 100 mM Water 1:5 24 hr DFP 10 mM Buffer (pH 6.4-7.2) 1:0.2 18 hr DFP 200 mM DMF 1:2 18 hr DFP pure — 1:25 1 hr DFP 10 mM Water 1:0.2 18 hr PMSF 10 mM Buffer (pH 5.0-8.0) 1:0.1 1 hr PMSF 100 mM DMSO 1:5 24 hr PMSF 100 mM Isopropanol 1:5 24 hr TLCK 1 mM Buffer (pH 5.0-8.0) 1:0.1 18 hr TLCK 5 mM DMSO 1:5 18 hr TLCK 5 mM Water 1:0.1 18 hr TPCK 1 mM Buffer (pH 5.0-8.0) 1:0.1 18 hr TPCK 1 mM DMSO 1:0.1 18 hr TPCK 1 mM Isopropanol 1:0.1 18 hr

Safe Use of Hazardous Chemicals A.1H.28

Supplement 58 Current Protocols in Molecular Biology 1. Prepare the modified Griess reagent by boiling 0.1 g α-naphthylamine in 20 ml water until it dissolves. While the solution is still hot, pour it into 150 ml of 15% aqueous acetic acid. Add 150 ml sulfanilic acid solution. This reagent should be stored at room temperature in a brown bottle. CAUTION: α-Naphthylamine is a carcinogen. 2. Add 3 ml of the reaction mixture treated to remove excess nitrite to 1 ml modified Griess reagent. Let stand 6 min at room temperature. 3. Measure the absorbance at 520 nm against a suitable blank.

DISPOSAL OF ENZYME INHIBITORS BASIC PROTOCOL 11 The enzyme inhibitors p-amidinophenylmethanesulfonyl fluoride (APMSF), 4-(2-ami- noethyl)benzenesulfonyl fluoride (AEBSF), phenylmethylsulfonyl fluoride (PMSF; Lunn and Sansone, 1994c), diisopropyl fluorophosphate (DFP; Lunn and Sansone, 1994d), Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK), and N-p-tosyl-L-phenylalan- ine chloromethyl ketone (TPCK; Lunn and Sansone, 1994c) may be degraded by reaction with 1 M NaOH. Destruction is >99.8% (except TPCK >98.3%). The exact reaction conditions depend on the solvent (see Table A.1H.8). The solutions that were decontami- nated are representative of those described in the literature. Materials Solutions of APMSF, AEBSF, PMSF, DFP, TLCK, or TPCK in buffer, DMSO, isopropanol, or water 1 M NaOH Glacial acetic acid Additional reagents and equipment for testing for the presence of the enzyme inhibitors (see Support Protocol 9) 1. If necessary, dilute the solutions with the same solvent so that the concentrations given in Table A.1H.8 are not exceeded. Bulk quantities of AEBSF, PMSF, and TPCK may be dissolved in isopropanol and bulk quantities of APMSF and TLCK may be dissolved in water at the concentrations shown in Table A.1H.8. Bulk quantities of DFP (a liquid) may be mixed directly with 1 M NaOH, taking care to make sure that all the DFP has mixed thoroughly, in the ratio shown in Table A.1H.8 (e.g., 40 ìl DFP with 1 ml of 1 M NaOH). 2. Add 1 M NaOH so that the ratio of solution to 1 M NaOH is that listed in Table A.1H.8. 3. Shake to ensure complete mixing, check that the solution is strongly basic (pH ≥12), and allow to stand for the time given in Table A.1H.8. 4. Neutralize the reaction mixture with acetic acid and test for the presence of residual enzyme inhibitor (see Support Protocol 9). 5. Discard the decontaminated reaction mixture.

Standard Measurements, Data, and Abbreviations A.1H.29

Current Protocols in Molecular Biology Supplement 58 Table A.1H.9 HPLC Conditions for Enzyme Inhibitors

Limit of Compound Mobile phase Detector Retention time detection AEBSF 40:60 (v/v) UV 225 nm 9.5 min 0.1 µg/ml acetonitrile:0.1% trifluoroacetic acid APMSF 40:60 (v/v) UV 232 nm 7.7 min 0.5 µg/ml acetonitrile:0.1% trifluoroacetic acid PMSF 50:50 (v/v) UV 220 nm 8 min 0.9 µg/ml acetonitrile:water TLCK 40:60 (v/v) UV 228 nm 9.5 min 0.37 µg/ml acetonitrile:0.1% trifluoroacetic acid TPCK 48:52 (v/v) acetonitrile:10 UV 228 nm 10.5 min 2 µg/ml mM pH 7 phosphate buffer

SUPPORT ANALYTICAL PROCEDURES TO DETECT ENZYME INHIBITORS PROTOCOL 9 DFP can be determined using a complex procedure involving the inhibition of chymotryp- sin activity. For more information, refer to Lunn and Sansone (1994d). A gas chroma- tographic method has also been described by Degenhardt-Langelaan and Kientz (1996). AEBSF, APMSF, PMSF, TLCK, and TPCK may be determined by reversed-phase HPLC (Snyder et al., 1997). The chromatographic conditions and limits of detection are shown in Table A.1H.9 (Lunn and Sansone, 1994c). Materials Decontaminated enzyme inhibitor solutions Acetonitrile (HPLC grade) Water (HPLC grade) 0.1% (v/v) trifluoroacetic acid in water 10 mM phosphate buffer, pH 7 250-mm × 4.6 mm-i.d. Microsorb C-8 reversed-phase HPLC column (Varian) or equivalent Additional reagents and equipment for reversed-phase liquid chromatography (Snyder et al., 1997) Analyze the decontaminated enzyme inhibitor solutions by reversed-phase HPLC using a 250-mm × 4.6-mm-i.d. Microsorb C-8 reversed-phase column, or equivalent, using the conditions shown in Table A.1H.9. In each case, the injection volume was 20 µl, the separation occurred at ambient temperature, and the flow rate was 1 ml/min. Check the analytical procedures by spiking an aliquot of the acidified reaction mixture with a small quantity of a dilute solution of the compound of interest.

Safe Use of Hazardous Chemicals A.1H.30

Supplement 58 Current Protocols in Molecular Biology REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2; for suppliers, see APPENDIX 4. Cyanide detection reagent Stir 3.0 g barbituric acid in 10 ml water. Add 15 ml of 4-methylpyridine and 3 ml concentrated HCl while continuing to stir. Cool and dilute to 50 ml with water. Store at room temperature. CAUTION: This reaction is exothermic. Decontamination solution Dissolve 4.2 g sodium nitrite (0.2 M final) and 20 ml hypophosphorous acid (3.3% w/v final) in 300 ml water. Prepare fresh. Horseradish peroxidase Dissolve hydrogen-peroxide oxidoreductase (EC 1.11.1.7 [Type II]; specific activity 150 to 200 purpurogallin U/mg, Sigma) in 1 g/liter sodium acetate to give 30 U/ml. Prepare fresh daily. For small-scale reactions, a more dilute solution can be used to avoid working with inconveniently small volumes. pH 5 buffer 2.04 g potassium hydrogen phthalate (0.05 M final) 38 ml 0.1 M potassium hydroxide (15 mM)

H2O to 200 ml Store at room temperature Phosphate buffer

13.6 g monobasic potassium phosphate (KH2PO4; 0.1 M final) 0.28 g dibasic sodium phosphate (Na2HPO4; 2 mM final) 3.0 g potassium bromide (KBr; 25 mM final)

1 liter H2O Store at room temperature Potassium bromide is necessary to make the assay for cyanide work correctly. Sodium azide indicator solution 0.1 g bromocresol purple (0.4% final) 18.5 ml 0.01 M potassium hydroxide (KOH; 7.4 mM final)

H2O to 25 ml Store at room temperature Sulfanilic acid solution Dissolve 0.5 g sulfanilic acid in 150 ml of 15% (v/v) aqueous acetic acid. Use immediately.

LITERATURE CITED Bretherick, L. (ed.) 1986. Hazards in the Chemical Laboratory, 4th ed. Royal Society of Chemistry, London. Bretherick, L., Urben, P.G., and Pitt, M.J. 1999. Bretherick’s Handbook of Reactive Chemical Hazards, 6th ed. Butterworth-Heinemann, London. Castegnaro, M., Barek, J., Dennis, J., Ellen, G., Klibanov, M., Lafontaine, M., Mitchum, R., van Roosmalen, P., Sansone, E.B., Sternson, L.A., and Vahl, M. (eds.) 1985. Laboratory Decontamination and Destruction of Carcinogens in Laboratory Wastes: Some Aromatic Amines and 4-Nitrobiphenyl. IARC Scientific Publications No. 64. International Agency for Research on Cancer, Lyon, France. Standard Cunniff, P. (ed.) 1995. Official Methods of Analysis of the Association of Official Analytical Chemists, 16th Measurements, ed., Ch. 4, p. 14. Association of Official Analytical Chemists, Arlington, Va. Data, and Abbreviations A.1H.31

Current Protocols in Molecular Biology Supplement 58 Degenhardt-Langelaan, C.E.A.M. and Kientz, C.E. 1996. Capillary gas chromatographic analysis of nerve agents using large volume injections. J. Chromatogr. A.723:210-214. Forsberg, K. and Keith, L.H. 1999. Chemical Protective Clothing Performance Index Book, 2nd ed. John Wiley & Sons, New York. Furr, A.K. (ed.) 2000. CRC Handbook of Laboratory Safety, 5th ed. CRC Press, Boca Raton, Fla. Lewis, R.J. Sr. 1999. Sax’s Dangerous Properties of Industrial Materials, 10th ed. John Wiley & Sons, New York. Lunn, G. and Sansone, E.B. 1985a. Destruction of cyanogen bromide and inorganic cyanides. Anal. Biochem. 147:245-250. Lunn, G. and Sansone, E.B. 1985b. Validation of techniques for the destruction of dimethyl sulfate. Am. Ind. Hyg. Assoc. J. 46:111-114. Lunn, G. and Sansone, E.B. 1987. Ethidium bromide: Destruction and decontamination of solutions. Anal. Biochem. 162:453-458. Lunn, G. and Sansone, E.B. 1989. Decontamination of ethidium bromide spills. Appl. Ind. Hyg. 4:234-237. Lunn, G. and Sansone, E.B. 1990a. Validated methods for degrading hazardous chemicals: Some alkylating agents and other compounds. J. Chem. Educ. 67:A249-A251. Lunn, G. and Sansone, E.B. 1990b. Degradation of ethidium bromide in alcohols. BioTechniques 8:372-373. Lunn, G. and Sansone, E.B. 1991a. The safe disposal of diaminobenzidine. Appl. Occup. Environ. Hyg. 6:49-53. Lunn, G. and Sansone, E.B. 1991b. Decontamination of aqueous solutions of biological stains. Biotech. Histochem. 66:307-315. Lunn, G. and Sansone, E.B. 1991c. Decontamination of ethidium bromide spills-author’s response. Appl. Occup. Environ. Hyg. 6:644-645. Lunn, G. and Sansone, E.B. 1994a. Destruction of Hazardous Chemicals in the Laboratory, 2nd ed. John Wiley & Sons, New York. Lunn, G. and Sansone, E.B. 1994b. Safe disposal of highly reactive chemicals. J. Chem. Educ. 71:972-976. Lunn, G. and Sansone, E.B. 1994c. Degradation and disposal of some enzyme inhibitors. Scientific note. Appl. Biochem. Biotechnol. 48:57-59. Lunn, G. and Sansone, E.B. 1994d. Safe disposal of diisopropyl fluorophosphate (DFP). Appl. Biochem. Biotechnol. 49:165-171. Lunn, G., Klausmeyer, P.K., and Sansone, E.B. 1994. Removal of biological stains from aqueous solution using a flow-through decontamination procedure. Biotech. Histochem. 69:45-54. Manufacturing Chemists Association. 1973. Laboratory Waste Disposal Manual. p. 136. Manufacturing Chemists Association, Washington, D.C. Mason, K.G. 1967. Hydrogen azide. In Mellor’s Comprehensive Treatise on Inorganic and Theoretical Chemistry, Vol. VIII (Suppl. II) pp. l-15. John Wiley & Sons, New York. National Research Council. 1983. Prudent Practices for Disposal of Chemicals from Laboratories, p. 88. National Academy Press, Washington, D.C. O’Neil, M.J. (ed.) 2001. The Merck Index, 13th ed. Merck & Co., Whitehouse Station, N.J. Patnode, W. and Wilcock, D.F. 1946. Methylpolysiloxanes. J. Am. Chem. Soc. 68:358-363. Shirakashi, T., Nakayama, K., Kakii, K., and Kuriyama, M. 1986. Removal of mercury from laboratory waste water with iron powder. Chem. Abstr. 105:213690y. Snyder, L.R., Kirkland, J.J., and Glajch, J.L. 1997. Practical HPLC Method Development, 2nd ed. John Wiley & Sons, New York.

KEY REFERENCES The following are good general references for laboratory safety.

American Chemical Society, Committee on Chemical Safety. 1995. Safety in Academic Chemistry Labora- tories, 6th ed. American Chemical Society, Washington, D.C. Castegnaro, M. and Sansone, E.B. 1986. Chemical Carcinogens. Springer-Verlag, New York. DiBerardinis, L.J., First, M.W., Gatwood, G.T., and Seth, A.K. 2001. Guidelines for Laboratory Design, Health and Safety Considerations, 3rd ed. John Wiley & Sons, New York. Fleming, D.D., Richardson, J.H., Tulis, J.J., and Vesley, D. 1995. Laboratory Safety, Principles and Practices, 2nd ed. American Society for Microbiology, Washington, D.C. Safe Use of Hazardous Freeman, N.T. and Whitehead, J. 1982. Introduction to Safety in the Chemical Laboratory. Academic Press, Chemicals San Diego. A.1H.32

Supplement 58 Current Protocols in Molecular Biology Fuscaldo, A.A., Erlick, B.J., and Hindman, B. (eds.) 1980. Laboratory Safety, Theory and Practice. Academic Press, San Diego. Lees, R. and Smith, A.F. (eds.) 1984. Design, Construction, and Refurbishment of Laboratories. Ellis Horwood, Chichester, United Kingdom. Montesano, R., Bartsch, H., Boyland, E., Della Porta, G., Fishbein, L., Griesemer, R.A., Swan, A.B., and Tomatis, L. (eds.) 1979. Handling Chemical Carcinogens in the Laboratory, Problems of Safety. IARC Scientific Publications No. 33. International Agency for Research on Cancer, Lyon, France. National Research Council. 1995. Prudent Practices in the Laboratory: Handling and Disposal of Chemicals. National Academy Press, Washington, D.C. Occupational Health and Safety. 1993. National Safety Council, Chicago. Pal, S.B. (ed.) 1991. Handbook of Laboratory Health and Safety Measures, 2nd ed. Kluwer Academic Publishers, Hingham, Mass. Rosenlund, S.J. 1987. The Chemical Laboratory: Its Design and Operation: A Practical Guide for Planners of Industrial, Medical, or Educational Facilities. Noyes Publishers, Park Ridge, N.J. Young, J.A. (ed.) 1991. Improving Safety in the Chemical Laboratory: A Practical Guide, 2nd ed. John Wiley & Sons, New York.

INTERNET RESOURCES http://www.ilpi.com/msds/index.html Where to find MSDSs on the internet. Contains links to general sites, government and nonprofit sites, chemical manufacturers and suppliers, pesticides, and miscellaneous sites. http://www.OSHA.gov OSHA web site. http://www.osha-slc.gov/OshStd_data/1910_1450.html Text of OSHA Standard 29 CFR 1910.1450: Occupational Exposure to Hazardous Chemicals in Laboratories. http://www.osha-slc.gov/OshStd_data/1910_1000_TABLE_Z-1.html Tables of permissible exposure limits (PELs) for air contaminants. http://www.osha-slc.gov/OshStd_data/1910_1000_TABLE_Z-2.html Tables of PELs for toxic and hazardous substances. http://hazard.com/msds/index.php Main site for Vermont SIRI. One of the best general sites to start a search. Browse manufacturers alphabeti- cally (for sheets not in the SIRI collection) or do a keyword search in the SIRI MSDS database. Lots of additional safety links and information. http://siri.uvm.edu/msds Alternate site for Vermont SIRI. http://tis.eh.doe.gov/docs/osh_tr/ch5.html DOE OSH technical reference chapter on personal protective equipment.

Contributed by George Lunn Baltimore, Maryland

Gretchen Lawler Purdue University West Lafayette, Indiana

Standard Measurements, Data, and Abbreviations A.1H.33

Current Protocols in Molecular Biology Supplement 58 Commonly Used Detergents APPENDIX 1I

Detergents are polar lipids that are soluble in water. The presence of both a hydrophobic and hydrophilic portion makes these compounds very useful for lysis of lipid membranes, solubilization of antigens, and washing of immune complexes.

TYPES OF DETERGENTS A large variety of detergents are available (Helenius et al., 1979). For biochemical studies, they are usually categorized according to the type of hydrophilic group they contain— anionic, cationic, amphoteric, or nonionic. Tables A.1I.1 and A.1I.2 list commonly used members of each type. In general, nonionic and amphoteric detergents are less denaturing for proteins than ionic detergents. Sodium cholate and sodium deoxycholate are the least denaturing of the commonly used ionic detergents. Two properties of detergents are important in their consideration for biological studies: the critical micelle concentration (CMC) and the micelle molecular weight (Table A.1I.1). The CMC is the concentration at which monomers of detergent molecules combine to form micelles; each detergent micelle has a characteristic micelle molecular weight. Detergents with a high micelle molecular weight, such as nonionic detergents, are difficult to remove from samples by dialysis. The CMC and the micelle molecular weight will vary depending on the buffer, salt concentration, pH, and temperature. In general, adding salt will lower the CMC and raise the micelle size.

Table A.1I.1 Physical Properties of Commonly Used Detergentsa,b

Molecular weight (Da) CMC Detergent mp (°C) Monomer Micelle % (w/v) M Anionic × −3 SDS 206 288 18,000 0.23 8.0 10 Cholate 201 430 4,300 0.60 1.4 × 10−2 Deoxycholate 175 432 4,200 0.21 5.0 × 10−3 Cationic −3 C16TAB 230 365 62,000 0.04 1.0 × 10 Amphoteric LysoPC — 495 92,000 0.0004 7.0 × 10−6 CHAPS157 615 6,150 0.49 1.4 × 10−3 Zwittergent 3-14 — 364 30,000 0.011 3.0 × 10−4 Nonionic Octyl glucoside 105 292 8,000 0.73 2.3 × 10−2 Digitonin 235 1,229 70,000 — — −5 C12E8 — 542 65,000 0.005 8.7 × 10 Lubrol PX — 582 64,000 0.006 1.0 × 10−4 Triton X-100 — 650 90,000 0.021 3.0 × 10−4 Nonidet P-40 — 603 90,000 0.017 3.0 × 10−4 Tween 80 — 1,310 76,000 0.002 1.2 × 10−5 aReprinted with permission from IRL Press (see Jones et al., 1987). bAbbreviations: C TAB, hexadecyl trimethylammonium bromide; CMC, critical micelle concentration; Standard 16 Measurements, LysoPC, lysophosphatidylcholine; mp, melting point; SDS, sodium dodecyl sulfate. Data, and Abbreviations Contributed by John E. Coligan A.1I.1 Current Protocols in Molecular Biology (1998) A.1I.1-A.1I.3 Copyright © 1998 by John Wiley & Sons, Inc. Supplement 43 Table A.1I.2 Chemical Properties of Commonly Used Detergentsa,b

Ionic detergents Nonionic detergents Property SDS CHO DOC C16 LYSCHA ZWI OGL DIG C 12 LUB TNX NP-40 T80 Strongly denaturingc +−−++/−−+/− −−−−− − − Dialyzable ++++−++/−+−−−−−− Ion exchangeabled ++++−−− −−−−− − − Complexes ions +++−−−− −−+/−+/−+/−+/−+/−

Strong A280 −−−−−−− −−−−+ + − Assay interference −−−−−−− −−−+/−+/−+/−+/− Cold precipitates +−++−−− −−−−− − − High cost −−−−+++ +++−− − − Availability +++++++/−+++/−+ + + + Toxicity −−−−−−− −−−−− − − Ease of purification +++++/−+ + − + − − − − − Radiolabeled +++−+−− +−+++ + + Defined composition +++++++ + −−− − − Auto-oxidation −−−−−−− − +++ + +

aAdapted from IRL Press (see Jones et al., 1987). b Abbreviations: C12, C12E8; C16, hexadecyl trimethylammonium bromide; CHA, CHAPS; CHO, cholate; DIG, digitonin; DOC, deoxycholate; LUB, lubrol PX; LYS, lysophosphatidylcholine; NP-40, Nonidet P-40; OGL, octyl glucoside; SDS, sodium dodecyl sulfate; T80, Tween 80; TNX, Triton X-100; ZWI, Zwittergent 3-14. c Denaturing refers to disruption of secondary and tertiary protein structure. dIonic detergents are unsuitable for ion-exchange chromatography (UNIT 10.10).

CHOICE OF DETERGENTS Ionic detergents are very good solubilizing agents, but they tend to denature proteins by destroying native three-dimensional structures. This denaturing ability is useful for SDS-PAGE (UNIT 10.2), but is detrimental where native structure is important, as when functional activities must be retained (antibody activity is usually retained in <0.1% SDS). Nonionic and mildly ionic detergents are less denaturing and can often be used to solubilize membrane proteins while retaining protein-protein interactions. The following detergent properties are detrimental in certain procedures: 1. Phenol-containing detergents (e.g., Triton X-100 and NP-40) have a high absorbance at 280 nm and hence interfere with protein monitoring during chromatography (most ionic detergents do not absorb at 280 nm; Brij- and Lubrol-series detergents are nonionic detergents that do not have substantial absorbance at 280 nm). Phenol-con- taining detergents also induce precipitation in the Folin protein assay (but they can be used with the Bradford protein assay; UNIT 10.1A). Finally, they are readily iodinated and so should not be present during radioiodination. 2. Many detergents have a very high micelle molecular weight (Table A.1I.1), which makes their use in gel filtration impossible since protein sizes are insignificant relative to the micelle size. In addition, such detergents cannot be readily removed by dialysis. 3. Sodium cholate and sodium deoxycholate are insoluble below pH 7.5 or above an ionic strength of 0.1%. SDS will often crystallize below 20°C. 4. Ionic detergents interfere with nondenaturing electrophoresis and isoelectric focus- Detergents ing. A.1I.2

Supplement 43 Current Protocols in Molecular Biology Detergents can be removed or exchanged for other detergents by a variety of procedures (Hjelmeland, 1979; Furth et al., 1984; Harlow and Lane, 1988). Ionic and amphoteric detergents can usually be removed by dialysis (APPENDIX 3C). Pierce makes Extracti-Gel D for removing a variety of detergents from protein solutions (Pierce Immunotechnology Catalog and Handbook on Protein Modification).

LITERATURE CITED Furth, A.J., Bolton, H., Potter, J., and Priddle, J.D. 1984. Separating detergents from proteins. Methods Enzymol. 104:318-328. Harlow, E. and Lane, D. 1988. Detergents. In Antibodies: A Laboratory Manual, pp. 687-689. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C. 1979. Properties of detergents. Methods Enzymol. 56:734-749. Hjelmand, L.M. 1979. Removal of detergents from membrane proteins. Methods Enzymol. 182:277-282. Jones, O.T., Earnest, J.P. and McNamee, M.G. 1987. Solubilization and reconstitution of membrane proteins. In Biological Membranes: A Practical Approach (J. Findlay and W.H. Evans, eds.), pp. 142-143. IRL Press, Oxford.

KEY REFERENCES Harlow and Lane, 1988. See above. Provides properties of commonly used detergents and means of removing them from proteins.

Hjelmeland, L.M. and Chrambach, A. 1984. Solubilization of functional membrane proteins. Methods Enzymol. 182:305-318. Describes properties of detergents and how to use them to solubilize proteins.

Johnstone, A. and Thorpe, R. 1982. Isolation and fractionation of lymphocytes. In Immunochemistry in Practice, pp. 94-101. Blackwell Scientific, Oxford. Provides details in use of detergents to solubilize cells and membranes.

Neugebaur, J.M. 1990. Detergents: An overview. Methods Enzymol. 182:239-282. Provides details on detergent properties and how to choose one for a particular application.

Contributed by John E. Coligan National Institute of Allergy and Infectious Diseases Bethesda, Maryland

Standard Measurements, Data, and Abbreviations A.1I.3

Current Protocols in Molecular Biology Supplement 43 Common Conversion Factors APPENDIX 1J

Table A.1J.1 lists some of the more common conversion factors for units of measure used throughout Current Protocols manuals, while Table A.1J.2 gives prefixes indicating powers of ten for SI units. Table A.1J.3 gives conversions between temperatures on the Celsius (Centigrade) and Fahrenheit scales. Celsius temperatures are converted to Fahrenheit temperatures by multiplying the Celsius figure by 9, dividing by 5, and adding 32, or by multiplying the Celsius figure by 1.8 and adding 32. Fahrenheit is converted to Celsius by subtracting 32 from the Fahrenheit figure, multiplying by 5, and dividing by 9. In Table A.1J.3, the center figure represents the temperature one has read on one of the scales; the figure to the left is the conversion of that figure into Celsius if read in Fahrenheit, while that to the right represents the conversion to Fahrenheit if read in Celsius: e.g., the temperature 88 Fahrenheit converts to 31.1°C, while the temperature 88 Celsius converts to 190.4°F.

Table A.1J.1 Unit of Measurement Conversion Chart

To convert: Into: Use the multiplier: amperes per square centimeter (amp/cm2) amperes per square inch (amp/in.2)6.452 amperes per square meter (amp/m2)104 amperes per square inch (amp/in.2) amperes per square centimeter (amp/cm2)0.1550 amperes per square meter (amp/m2)1.55 × 103 ampere-hours (amp-hr) coulombs (C) 3.6 × 103 faradays 3.731 × 10−2 atmospheres (atm) bar 1.01325 millimeters of mercury (mmHg) or torr 760 tons per square foot (tons/ft2)1.058 bar atmospheres (atm) 0.9869 dynes per square centimeter (dyn/cm2)106 kilograms per square meter (kg/m2)1.020 × 104 pounds per square foot (lb/ft2)2,089 pounds per square inch (lb/in.2 or psi) 14.50 British thermal units (Btu) ergs 1.0550 × 1010 gram-calories (g-cal) 252.0 horsepower-hours (hp-hr) 3.931 × 104 joules (J) 1,054.8 kilogram-calories (kg-cal) 0.2520 kilogram-meters (kg-m) 107.5 kilowatt-hours (kW-hr) 2.928 × 10−4 British thermal unit per minute (Btu/min) foot-pounds per second (ft-lb/sec) 12.96 horsepower (hp) 2.356 × 10−2 watts (W) 17.57 bushels cubic feet (ft3)1.2445 cubic inches (in.3) 2,150.4 cubic meters (m3)3.524 × 10−2 liters 35.24 quarts, dry 32.0 continued

Current Protocols in Molecular Biology (2000) A.1J.1-A.1J.8 A.1J.1 Copyright © 2000 by John Wiley & Sons, Inc. Supplement 49 Table A.1J.1 Unit of Measurement Conversion Chart, continued

To convert: Into: Use the multiplier: degrees Celsius or Centigrade (°C) degrees Fahrenheit (°F) (°C × 9⁄5) + 32 Kelvin (K) (°C) + 273.15 degree Fahrenheit (°F) degrees Celsius (°C) 5⁄9 × (°F − 32) Kelvin (K) [5⁄9 × (°F − 32)] + 273.15 centimeters (cm) feet (ft) 3.281 × 10−2 inches (in.) 0.3937 kilometers (km) 10−5 meters (m) 10−2 miles 6.214 × 10−6 millimeters (mm) 10.0 mils 393.7 yards 1.094 × 10−2 centimeters per second (cm/sec) feet per minute (ft/min) 1.1969 feet per second (ft/sec) 3.281 × 10−2 kilometers per hour (km/hr) 3.6 × 10−2 meters per minute (m/min) 0.6 miles per hour (miles/hr) 2.237 × 10−2 miles per minute (miles/min) 3.728 × 10−4 coulombs (C) faradays 1.036 × 10−5 coulombs per square centimeter (C/cm2) coulombs per square inch (C/in.2) 64.52 coulombs per square meter (C/m2)104 coulombs per square inch (C/in.2) coulombs per square centimeter (C/cm2)0.1550 coulombs per square meter (C/m2)1.55 × 103 cubic centimeters (cm3) cubic feet (ft3)3.531 × 10−5 cubic inches (in.3)6.102 × 10−2 cubic meters (m3)10−6 cubic yards 1.308 × 10−6 gallons, U.S. liquid 2.642 × 10−4 liters 10−3 pints, U.S. liquid 2.113 ×10−3 quarts, U.S. liquid 1.057 × 10−3 days hours (hr) 24.0 minutes (min) 1.44 × 103 seconds (sec) 8.64 × 104 degrees (of angle; °) minutes (min) 60.0 quadrants, of angle 1.111 × 10−2 radians (rad) 1.745 × 10−2 seconds (sec) 3.6 × 104 drams grams (g) 1.7718 grains 27.3437 ounces, avoirdupois (oz) 6.25 × 10−2 dynes (dyn) joules per centimeter (J/cm) 10−7 joules per meter (J/m) or newtons (N) 10−5 kilograms (kg) 1.020 × 10−6 pounds (lb) 2.248 × 10−6

continued A.1J.2

Supplement 49 Current Protocols in Molecular Biology Table A.1J.1 Unit of Measurement Conversion Chart, continued

To convert: Into: Use the multiplier: faradays ampere-hours (amp-hr) 26.80 coulombs (C) 9.649 × 10−4 foot-pounds per minute (ft-lb/min) British thermal units per minute (Btu/min) 1.286 × 10−3 foot-pounds per second (ft-lb/sec) 1.667 × 10−2 horsepower (hp) 3.030 × 10−5 kilogram-calories per minute (kg-cal/min) 3.24 × 10−4 kilowatts (kW) 2.260 × 10−5 grams (g) decigrams (dg) 10 dekagrams (Dg) 0.1 dynes (dyn) 980.7 grains 15.43 hectograms (hg) 10−2 kilograms (kg) 10−3 micrograms (µg) 106 milligrams (mg) 103 ounces, avoirdupois (oz) 3.527 × 10−2 ounces, troy 3.215 × 10−2 pounds (lb) 2.205 × 10−3 horsepower (hp) horsepower, metric 1.014 inches (in.) centimeters (cm) 2.540 feet (ft) 8.333 × 10−2 meters (m) 2.540 × 10−2 miles 1.578 × 10−5 millimeters (mm) 25.40 yards 2.778 × 10−2 inches of mercury (in. Hg) atmospheres (atm) 3.342 × 10−2 kilogram per square centimeter (kg/cm2)3.453 × 10−2 kilograms per square meter (kg/m2)345.3 pounds per square foot (lb/ft2) 70.73 pounds per square inch (lb/in.2 or psi) 0.4912 joules (J) British thermal units (Btu) 9.480 × 10−4 ergs 107 foot-pounds (ft-lb) 0.7376 kilogram-calories (kg-cal) 2.389 × 10−4 kilogram-meters (kg-m) 0.1020 newton-meter (N-m) 1 watt-hours (W-hr) 2.778 × 10−4 Kelvin (K) degrees Celsius (°C) K − 273.13 degrees Fahrenheit (°F) [(K − 273.13) × 9⁄5] + 32 kilolines maxwells (Mx) 103 kilometers (km) centimeters (cm) 105 feet (ft) 3,281 inches (in.) 3.937 × 104 meters (m) 103 miles 0.6214 yards 1,094 continued

A.1J.3

Current Protocols in Molecular Biology Supplement 49 Table A.1J.1 Unit of Measurement Conversion Chart, continued

To convert: Into: Use the multiplier: kilowatts (kW) British thermal units per minute (Btu/min) 56.92 foot-pounds per minute (ft-lb/min) 4.426 × 104 horsepower (hp) 1.341 kilogram-calories per minute (kg-cal/min) 14.34 liters bushels, U.S. dry 2.838 × 10−2 cubic centimeters (cm3)103 cubic feet (ft3)3.531 × 10−2 cubic inches (in.3) 61.02 cubic meters (m3)10−3 cubic yards 1.308 × 10−3 gallons, U.S. liquid 0.2642 gallons, imperial 0.21997 kiloliter (kl) 10−3 pints, U.S. liquid 2.113 quarts, U.S. liquid 1.057 maxwells (Mx) webers (W) 10−8 micrograms (µg) grams (g) 10−6 microliters (µl) liters 10−6 milligrams (mg) grams (g) 10−3 milligrams per liter (mg/liter) parts per million (ppm) 1.0 millihenries (mH) henries (H) 10−3 milliliters (ml) liters 10−3 millimeters (mm) centimeters (cm) 0.1 feet (ft) 3.281 × 10−3 inches (in.) 3.937 × 10−2 kilometers (km) 10−6 meters (m) 10−3 miles 6.214 × 10−7 millimeters of mercury (mmHg) or torr atmospheres (atm) 1.316 × 10−3 kilograms per square meter (kg/m2)136.0 pounds per square foot (lb/ft2) 27.85 pounds per square inch (lb/in.2 or psi) 0.1934 nepers (Np) decibels (dB) 8.686 newtons (N) dynes (dyn) 105 kilograms, force (kg) 0.10197162 pounds, force (lb) 4.6246 × 10−2 ohms (Ω)megaohms (MΩ)106 microhms (µΩ)10−6 ounces, avoirdupois drams 16.0 grains 437.5 grams (g) 28.349527 pounds (lb) 6.25 × 10−2 ounces, troy 0.9115 tons, metric 2.835 × 10−5

continued

A.1J.4

Supplement 49 Current Protocols in Molecular Biology Table A.1J.1 Unit of Measurement Conversion Chart, continued

To convert: Into: Use the multiplier: ounces, fluid cubic inches (in3)1.805 liters 2.957 × 10−2 ounces, troy grains 480.0 grams (g) 31.103481 ounces, avoirdupois (oz) 1.09714 pounds, troy 8.333 × 10−2 pascal (P) newton per square meter (N/m2)1 pounds, force (lb) newtons (N) 21.6237 pounds per square foot (lb/ft2) atmospheres (atm) 4.725 × 10−4 inches of mercury (in. Hg) 1.414 × 10−2 kilograms per square meter (kg/m2)4.882 pounds per square inch (lb/in2 or psi) 6.944 × 10−3 pounds per square inch (lb/in.2 or psi) atmospheres (atm) 6.804 × 10−2 inches of mercury (in. Hg) 2.036 kilograms per square meter (kg/m2)703.1 pounds per square foot (lb/ft2)144.0 bar 6.8966 × 10−2 quadrants, of angle degrees (°)90.0 minutes (min) 5.4 × 103 radians (rad) 1.571 seconds (sec) 3.24 × 105 quarts, dry cubic inches (in.3) 67.20 quarts, liquid cubic centimeters (cm3)946.4 cubic feet (ft3)3.342 × 10−2 cubic inches (in.3) 57.75 cubic meters (m3)9.464 × 10−4 cubic yards 1.238 × 10−3 gallons 0.25 liters 0.9463 radians (rad) degrees (°) 57.30 minutes (min) 3,438 quadrants 0.6366 seconds (sec) 2.063 × 105 torr see millimeter of mercury watts (W) British thermal units per hour (Btu/hr) 3.413 British thermal units per min (Btu/min) 5.688 × 10−2 ergs per second (ergs/sec) 107 webers (Wb) maxwells (M) 108 kilolines 105

Useful Data A.1J.5

Current Protocols in Molecular Biology Supplement 49 Table A.1J.2 Power of Ten Prefixes for SI Units

Prefix Factor Abbreviation atto 10−18 a femto 10−15 f pico 10−12 p nano 10−9 n micro 10−6 µ milli 10−3 m centi 10−2 c deci 10−1 d deca 101 da hecto 102 h kilo 103 k myria 104 my mega 106 M giga 109 G tera 1012 T peta 1015 P exa 1018 E

Common Conversion Factors A.1J.6

Supplement 49 Current Protocols in Molecular Biology Table A.1J.3 Celsius/Fahrenheit Temperature Conversion Chart

Degrees Celsius (°C) Temperature Degrees Fahrenheit (°F) −17.8 0 32.0 −17.2 1 33.8 −16.7 2 35.6 −16.1 3 37.4 −15.6 4 39.2 −15.0 5 41.0 −14.4 6 42.8 −13.9 7 44.6 −13.3 8 46.4 −12.8 9 48.2 −12.2 10 50.0 −11.7 11 51.8 −11.1 12 53.6 −10.6 13 55.4 −10.0 14 57.2 −9.4 15 59.0 −8.9 16 60.8 −8.3 17 62.6 −7.8 18 64.4 −7.2 19 66.2 −6.7 20 68.0 −6.1 21 69.8 −5.6 22 71.6 −5.0 23 73.4 −4.4 24 75.2 −3.9 25 77.0 −3.3 26 78.8 −2.8 27 80.6 −2.2 28 82.4 −1.7 29 84.2 −1.1 30 86.0 −0.6 31 87.8 0.0 32 89.6 0.6 33 91.4 1.1 34 93.2 1.7 35 95.0 2.2 36 96.8 2.8 37 98.6 3.3 38 100.4 3.9 39 102.2 4.4 40 104.0 5.0 41 105.8 5.6 42 107.6 6.1 43 109.4 6.7 44 111.2 7.2 45 113.0 7.8 46 114.8 8.3 47 116.6 8.9 48 118.4 9.4 49 120.2 10.0 50 122.0 10.6 51 123.8 continued Useful Data A.1J.7

Current Protocols in Molecular Biology Supplement 49 Table A.1J.3 Celsius/Fahrenheit Temperature Conversion Chart, continued

Degrees Celsius (°C) Temperature Degrees Fahrenheit (°F)

11.1 52 125.6 11.7 53 127.4 12.2 54 129.2 12.8 55 131.0 13.3 56 132.8 13.9 57 134.6 14.4 58 136.4 15.0 59 138.2 15.6 60 140.0 16.1 61 141.8 16.7 62 143.6 17.2 63 145.4 17.8 64 147.2 18.3 65 149.0 18.9 66 150.8 19.4 67 152.6 20.0 68 154.4 20.6 69 156.2 21.1 70 158.0 21.7 71 159.8 22.2 72 161.6 22.8 73 163.4 23.3 74 165.2 23.9 75 167.0 24.4 76 168.8 25.0 77 170.6 25.6 78 172.4 26.1 79 174.2 26.7 80 176.0 27.2 81 177.8 27.8 82 179.6 28.3 83 181.4 28.9 84 183.2 29.4 85 185.0 30.0 86 186.8 30.6 87 188.6 31.1 88 190.4 31.7 89 192.2 32.2 90 194.0 32.8 91 195.8 33.3 92 197.6 33.9 93 199.4 34.4 94 201.2 35.0 95 203.0 35.6 96 204.8 36.1 97 206.8 36.7 98 208.4 37.2 99 210.2 37.8 100 212.0

Common Conversion Factors A.1J.8

Supplement 49 Current Protocols in Molecular Biology Compendium of Drugs Commonly Used in APPENDIX 1K Molecular Biology Research

The following appendix includes an alphabetical list of drugs commonly used to examine various biological processes. Table A.1K.1 lists the drugs by activity and provides recent references. Indicated under each drug listed is its mode of action, generally including several specific experimental examples; solvent(s) used to solubilize the drug; stock and working concentrations or ranges; storage conditions; and duration of incubation with cells to achieve the desired effects. Except where indicated, the majority of drugs in this list are cell-permeant. However, despite the well characterized selectivity of many of the following drugs in vitro, the corresponding effects upon their intracellular targets may not be precisely determined directly by their extracellular concentrations, since their cell-permeation properties are not known. Therefore, several different concentrations of any particular drug, as well as alternative methods of determining drug selectivity, should be examined. Several of these drugs are members of large families, such as those targeting protein kinases and phosphatases, as well as those that affect intracellular Ca2+ levels. Many of these family members have different selectivities and potencies toward similar targets, and a complete listing is not included here. The reader may consult catalogs from the following companies, which have several of these family members available: Sigma, Alexis Biochemicals (including LC Laboratories), Calbiochem, Biomol, Molecular Probes, Boehringer Mannheim, Oxford Glycosystems, and Avanti Polar Lipids. Although not specifically indicated, many of the following drugs are hazardous and should be handled with extreme care. Material Safety Data Sheets (MSDSs) are often provided for products that are hazardous or toxic. In some cases, these products are new and have not been tested for toxicity. Thus, care should be taken to ensure the safe handling of all products.

Table A.1K.1 Biological Activity of Drugs Commonly Used In Molecular Biological Research

Drug References DNA replication inhibitors Aphidicolin Borner et al., 1995. Cancer Res. 55:2122-2128; Debec et al., 1996. J. Cell Biol. 134:103-115; Jackson et al., 1995. J. Cell Biol. 130:755- 769; Urbani et al., 1995. Exp. Cell Res. 219:159-168 Ara-C Grant et al., 1994. Oncol. Res. 6:87-99; Tomkins et al., 1994. J. Cell Sci. 107:1499-1507 Camptothecin Carettoni et al., 1994. Biochem. J. 299:623-629; Desai et al., 1997. JBC 272:24159-24164; Li et al., 1994. JBC 269:7051-7054 Etoposide Kaufmann et al., 1993. Cancer Res. 53:3976-3985; Meyer et al., 1997. J. Cell Biol. 136:775-788; Shao et al., 1997. JBC 272:31321-31325; Terada et al., 1993. J. Med. Chem. 36:1689-1699; Wozniak et al., 1991. J. Clin. Oncol. 9:70-76 Hydroxyurea Anand et al., 1995. Cancer Lett. 88:101-105; Nishijima et al., 1997. J. Cell Biol. 138:1105-1116; Oliver et al., 1997. JBC 272:10624-10630 L-Mimosine Gilbert et al., 1995. JBC 270:9597-9606; Lin et al., 1996. JBC 271:2548-2556; Park et al., 1997. J. Neurosci. 17:1256-1270 Standard continued Measurements, Data, and Abbreviations Contributed by Nelson B. Cole A.1K.1 Current Protocols in Molecular Biology (2002) A1.K.1-A1.K.26 Copyright © 2002 by John Wiley & Sons, Inc. Supplement 59 Table A.1K.1 Biological Activity of Drugs Commonly Used In Molecular Biological Research, continued Drug References Drugs affecting the cytoskeleton Colcemid Barlow et al., 1994. J. Cell Biol. 126:1017-1029; Bonfoco et al., 1995. Exp. Cell Res. 218:189-200; Goswami et al., 1994. Exp. Cell Res. 214:198-208 Colchicine Bonfoco et al., 1995. Exp. Cell Res. 218:189-200; Lindenboim et al., 1995. J. Neurochem. 64:1054-1063 Cytochalasin B Benya and Padilla, 1993. Exp. Cell Res. 204:268-277; Takeshita et al., 1998. Cancer Lett. 126:75-81; Tanaka et al., 1994. Exp. Cell Res. 213:242-252 Cytochalasin D Cooper, 1987. J. Cell Biol. 105:1473-1478; Radhakrishna and Donaldson, 1997. J. Cell Biol. 139:49-61; Sasaki et al., 1995. PNAS 92:2026-2030; Wang et al., 1994. Am. J. Physiol. 267:F592-F598 Latrunculins Wada et al., 1998. J. Biochem. 123:946-952; Lamaze et al., 1997. JBC 272:20332-20335; Spector et al., 1989. Cell Motil. Cytoskeleton 13:127-144 Nocodazole Cole et al., 1996. Mol. Biol Cell. 7:631-650; Liao et al., 1995. J. Cell Sci. 108:3473-3483; Vasquez et al., 1997. Mol. Biol. Cell 8:973-985 Taxol Derry et al., 1995. Biochemistry 34:2203-2211; Ding et al., 1990. Science 248:370-372; Gallo, 1998. J. Neurobiol. 35:121-140; Jordan et al., 1993. PNAS 90:9552-9556; Mogensen and Tucker, 1990. J. Cell Sci. 97:101-107 Vinblastine Dhamodharan et al., 1995. Mol. Biol. Cell 6:1215-1229; Lobert et al., 1998. Cell Motil. Cytoskeleton 39:107-121; Panda et al., 1996. JBC 271:29807-29812; Rai and Wolff, 1996. JBC 271:14707-14711; Taki et al., 1998. J. Neurooncol. 36:41-53; Tsukidate et al., 1993. Am. J. Pathol. 143:918-925 Drugs affecting intracellular Ca2+ A23187 Elia et al., 1996. JBC 27:16111-16118; Kao, 1994. Methods Cell Biol. 40:155-181; Kao et al., 1990. J. Cell Biol. 111:183-196 BAPTA Bissonnette et al., 1994. Am. J. Physiol. 267:G465-G475; Smith et al., 1992. Biochem. J. 288:925-929; Tsien, 1980. Biochemistry 19:2396- 2404 Ionomycin Aagaard-Tillery and Jelinek, 1995. J. Immunol. 155:3297-3307; Rock et al., 1997. JBC 272:33377-33383; Stewart et al., 1998. J. Cell Biol. 140:699-707 Thapsigargin Kuznetsov et al., 1993. JBC 268:2001-2008; Lodish et al., 1992. JBC 267:12753-12760; Takemura et al., 1989. JBC 264:12266-12271; Won and Orth, 1995. Endocrinology 136:5399-5408; Wong et al., 1993. Biochem. J. 289:71-79 Drugs affecting oligosaccharide biosynthesis/processing Castanospermine Ahmed et al., 1995. Biochem. Biophys. Res. Commun. 208:267-273; Bass et al., 1998. J. Cell Biol. 141:637-646; Gruters et al., 1987. Nature 330:74-77; Martina et al., 1998. JBC 273:3725-3731 Deoxymannojirimycin Elbein et al., 1984. Arch. Biochem. Biophys. 235:579-588; McDowell et al., 1987. Virology 161:37-44; Slusarewicz and Warren, 1995. Glycobiology 5:154-155; Wojczyk et al., 1998. Glycobiology 8:121-130 Deoxynojirimycin Labriola et al., 1995. J. Cell Biol. 130:771-779; Martina et al., 1998. JBC 273:3725-3731; Romero et al., 1985. Biochem. J. 226:733-740; Schlesinger et al., 1984. JBC 259:7597-7601 Compendium of Drugs Commonly Tunicamycin Bush et al., 1994. Biochem. J. 303:705-708; Kuznetsov et al., 1997. Used in JBC 272:3057-3063; Lodish and Kong, 1984. J. Cell Biol. 98:1720- Molecular 1729 Biology Research continued A.1K.2

Supplement 59 Current Protocols in Molecular Biology Table A.1K.1 Biological Activity of Drugs Commonly Used In Molecular Biological Research, continued Drug References Drugs affecting the pH of intracellular organelles Ammonium chloride Breuer et al., 1995. JBC 270:24209-24215; Davis and Mecham, 1996. JBC 271:3787-3794; Smith et al., 1997. JBC 272:5640-5646

Bafilomycin A1 Bowman et al., 1988. PNAS 85:7972-7976; Calvert and Sanders, 1995. JBC 270:7272-7280; Furuchi et al., 1993. JBC 268:27345-27348 CCCP Arai et al., 1996. Biochem. Biophys. Res. Commun. 227:433-439; Babcock et al., 1997. J. Cell Biol. 136:833-844; Kao, 1994. Methods Cell Biol. 40:155-181; Simpson and Russell, 1996. JBC 271:33493- 33501 Chloroquine Claus et al., 1998. JBC 273:9842-9851; Garcia-Sainz and Mendoza- Mendoza, 1998. Eur. J. Pharmacol. 342:333-338; Passos and Garcia, 1998. Biochem. Biophys. Res. Commun. 245:155-160; Wunsch et al., 1998. J. Cell Biol. 140:335-345 Concanamycin B Akifusa et al., 1998. Exp. Cell Res. 238:82-89; Nishihara et al., 1995. Biochem. Biophys. Res. Commun. 212:255-262; Yilla et al., 1993. JBC 268:19092-19100 Monensin Griffiths et al., 1983. J. Cell Biol. 96:835-850; Hardy et al., 1997. JBC 272:6812-6817; Kallen et al., 1993. Biochim. Biophys. Acta 1166:305- 308; Shiao and Vance, 1993. JBC 268:26085-26092 Nigericin Sandvig et al., 1989. Methods Cell Biol. 32:365-382; Scorrano et al., 1997. JBC 272:12295-12299; Vercesi et al., 1993. JBC 268:8564-8568 Drugs that lead to increased intracellular cAMP levels 8-Bromo–cyclic AMP Boyer and Thiery, 1993. J. Cell Biol. 120:767-776; Hei et al., 1991. Mol. Pharmacol. 39:233-238; Sandberg et al., 1991. Biochem. J. 279:521-527 Cholera toxin Hansen and Casanova, 1994. J. Cell Biol. 126:677-687; Lencer et al., 1995. J. Cell Biol. 131:951-962; Ma and Weiss, 1995. Methods Cell Biol. 49:471-485; Moss and Vaughan, 1992. Curr. Top. Cell. Regul. 32:49-72 Dibutyryl cyclic AMP Cong et al., 1998. JBC 273:660-666; O’Malley et al., 1997. J. Cell Biol. 138:159-165; Sandvig et al., 1994. J. Cell Biol. 126:53-64; Yuan et al., 1996. JBC 271:27090-27098 Forskolin Galli et al., 1995. J. Neurosci. 15:1172-1179; Lippincott-Schwartz et al., 1991. J. Cell Biol. 112:567-577; Laurenza et al., 1989. Trends Pharmacol. Sci. 10:442-447; Nickel et al., 1996. JBC 271:15870- 15873 Kinase inhibitors Bisindolylmaleimide I Das and White, 1997. JBC 272:14914-14920; Toullec et al., 1991. (GF 109203 X) JBC 266:15771-15781; Uberall et al., 1997. JBC 272:4072-4078 Calphostin C Dubyak and Kertesy, 1997. Arch. Biochem. Biophys. 341:129-139; Hartzell and Rinderknecht, 1996. Am. J Physiol. 270:C1293-C1299; Jarvis et al., 1994. Cancer Res. 54:1707-1714 Chelerythrine chloride Barg et al., 1992. J. Neurochem. 59:1145-1152; Herbert et al., 1990. Biochem. Biophys. Res. Commun. 172:993-999; Jarvis et al., 1994. Cancer Res. 54:1707-1714; Jia et al., 1997. JBC 272:4978-4984; Kandasamy et al., 1995. JBC 270:29209-29216 Genistein Akiyama et al., 1987. JBC 262:5592-5595; Chen et al., 1997. JBC 272:27401-27410; Kranenburg et al., 1997. J. Cell Sci. 110:2417-2427 H-7 Barria et al., 1997. Science 276:2042-2045; Kawamoto and Hidaka, 1984. Biochem. Biophys. Res. Commun. 125:258-264; Wang et al., Standard 1997. JBC 272:1817-1821 Measurements, Data, and continued Abbreviations A.1K.3

Current Protocols in Molecular Biology Supplement 59 Table A.1K.1 Biological Activity of Drugs Commonly Used In Molecular Biological Research, continued Drug References Herbimycin A Cowen et al., 1996. JBC 271:22297-22300; Fukazawa et al., 1991. Biochem. Pharmacol. 42:1661-1671; Tiruppathi et al., 1997. JBC 272:25968-25975 KN-62 Bouvard et al., 1998. J. Cell Sci. 111:657-665; Enslen and Soderling, 1994. JBC 269:20872-20877; Wang Hy et al., 1997. JBC 272:1817- 1821; Doroudchi et al., 1997. J. Neurosci. Res. 50:514-521. LY294002 Vlahos et al., 1994. JBC 269:5241-5248; Vlahos et al., 1995. J. Immunol. 154:2413-2422 ML-7 Ohkubo et al., 1996. Eur. J. Pharmacol. 298:175-183; Saitoh et al., 1987. JBC 262:7796-7801; Watanabe et al., 1998. FASEB J. 12:341-348 Olomoucine Abraham et al., 1995. Biol. Cell 83:105-120; Glab et al., 1994. FEBS Lett. 353:207-211; Howell et al., 1997. Cell. Motil. Cytoskeleton 38:201-214; Misteli and Warren, 1995. J. Cell Sci. 108:2715-2727 PD 98059 Acharya et al., 1998. Cell 92:183-192; Alessi et al., 1995. JBC 270:27489-27494; Dudley et al., 1995. PNAS 92:7686-7689; Karpova et al., 1997. Am. J. Physiol. 272:L558-L565; Waters et al., 1995. JBC 270:20883-20886 Piceatannol Keely and Parise, 1996. JBC 271:26668-26676; Oliver et al., 1994. JBC 269:29697-29703; Peters et al., 1996. JBC 271:4755-4762 Staurosporine Couldwell et al., 1994. FEBS Lett. 345: 43-46; Kiss and Deli, 1992. Biochem. J. 288: 853-858; Janicke et al., 1998. JBC 273:9357-9360; Orr et al., 1998. JBC 273:3803-3807 Tyrphostins Antonyak et al., 1998. JBC 273:2817-2822; Austin and Shields, 1996. J. Cell Biol. 135:1471-1483; Gazit et al., 1989. J. Med. Chem. 32:2344-2352; Gohla et al., 1998. JBC 273:4653-4659 Wortmannin Cross et al., 1995. JBC 270:25352-25355; Goeger et al., 1988. Biochem. Pharmacol. 37:978-981; Jones and Howell, 1997. J. Cell Biol. 139:339-349; Ptasznik et al., 1997. J. Cell Biol. 137:1127-1136 Phosphatase inhibitors Calyculin A Ishihara et al., 1989. Biochem. Biophys. Res. Commun. 159:871-877; Murakami et al., 1994. Neurosci. Lett. 176: 181-184; Takeuchi et al., 1994. Biochem. Biophys. Res. Commun. 205:1803-1807 Microcystin-LR Bagu et al., 1997. JBC 272:5087-5097; Eriksson et al., 1990. Biochim. Biophys. Acta 1025:60-66; Honkanen et al., 1990. JBC 265:19401- 19404; Rabouille et al., 1995. J. Cell Biol. 129:605-618; Toivola et al., 1997. J. Cell Sci. 110:23-33 Okadaic acid Gjertsen et al., 1994. J. Cell Sci. 107:3363-3377; Haystead et al., 1989. Nature 337:78-81; Kiguchi et al., 1994. Cell Growth Differ. 5:995-1004; Lucocq, 1992. J. Cell Sci. 103:875-880; Ohoka et al., 1993. Biochem. Biophys. Res. Commun. 197:916-921; Suganuma et al., 1988. PNAS 85:1768-1771 Takai et al., 1987. FEBS Lett. 217:81-84 Phenylarsine oxide Han and Kohanski, 1997. Biochem. Biophys. Res. Commun. 239:316- 321; Fleming et al., 1996. JBC 271:11009-11015; Krutetskaia et al., 1997. Tsitologiya 39:1116-1130; Kussmann and Przybylski, 1995. Methods Enzymol. 251:430-435 Sodium orthovanadate Brown and Gordon, 1984. JBC 259:9580-9586; Cook et al., 1997. JBC 272:13309-13319; Griffith et al., 1998. JBC 273:10771-10776; Munoz et al., 1992. JBC 267:10381-10388; Seedorf et al., 1995. JBC 270:18953-18960; Seglen and Gordon, 1981. JBC 256:7699-7701; Compendium of Swarup et al., 1982. Biochem. Biophys. Res. Commun. 107:1104-1109 Drugs Commonly Used in continued Molecular Biology Research A.1K.4 Protease inhibitors

Supplement 59 Current Protocols in Molecular Biology Table A.1K.1 Biological Activity of Drugs Commonly Used In Molecular Biological Research, continued Drug References Calpain inhibitor I Figueiredo-Pereira et al., 1994. J. Neurochem. 62: 1989-1994; Klafki et al., 1995. Neurosci Lett. 201:29-32; Milligan et al., 1996. Arch. Biochem. Biophys. 335:388-395 E-64 Banik et al., 1997. Brain Res. 1997 748:205-210; Bush et al., 1997. JBC 272:9086-9092; Sarin et al., 1994. J. Immunol. 153:862-872 Lactacystin Choi et al., 1997. JBC 272:28479-28484; Dick et al., 1996. JBC 271:7273-7276; Fenteany et al., 1995. Science 268:726-731; Kim et al., 1997. JBC 272:11006-11010; Oda et al., 1996. Biochem. Biophys. Res. Commun. 219: 800-805 Leupeptin Montenez et al., 1994. Toxicol. Lett. 73:201-208; Sarin et al., 1994. J. Immunol. 153:862-872; Wang et al., 1995. JBC 270:24924-24931 MG-132 Jensen et al., 1995. Cell 83:129-135; Lee and Goldberg, 1996. JBC 271:27280-27284; Meerovitch et al., 1997. JBC 272:6706-6713; Salceda and Caro, 1997. JBC 272:22642-22647 PMSF Darby et al., 1998. Biochemistry 37:783-791; Turini et al., 1969. J. Pharmacol. Exp. Ther. 167:98-104; Weaver et al., 1993. Biochem. Cell. Biol. 71:488-500 Pepstatin A Bode and Huber, 1992. Eur. J. Biochem. 204:433-451; Shields et al., 1991. Biochem. Biophys. Res. Commun. 177:1006-1012; Simon et al., 1995. Biochim. Biophys. Acta 1268: 143-151; Yamada et al., 1996. J. Immunol. 157:901-907; Wang et al., 1995. JBC 270:24924-24931 Protein synthesis inhibitors Anisomycin Dong Chen et al., 1996. JBC 271:6328-6332; Kardalinou et al., 1994. Mol. Cell. Biol. 14:1066-1074; Sidhu and Omiecinski, 1998. JBC 273:4769-4775 Cycloheximide Chow et al., 1995. Exp. Cell Res. 216:149-159; Cotter et al., 1992. Anticancer Res. 12:773-779; Waring, 1990. JBC 265:14476-14480 Emetine Burhans et al., 1991. EMBO J. 10:4351-4360; Gabathuler et al., 1998. J. Cell Biol. 140:17-27; Sidhu and Omiecinski, 1998. JBC 273:4769- 4775 Hygromycin Gaken et al., 1992. Biotechniques 13:32-34; Hamada et al., 1994. Curr. Genet. 26:251-255; Lama and Carrasco, 1992. JBC 267:15932- 15937 Puromycin Chow et al., 1995. Exp. Cell Res. 216:149-159; Kislauskis et al., 1997. J. Cell Biol. 136:1263-1270; Tachibana et al., 1997. EMBO J. 16:4333- 4339; Zhang et al., 1997. Mol. Biol. Cell 8:1943-1954 Transcription inhibitors Actinomycin D Kuhn and Henderson, 1995. JBC 270:20509-20515; McGary et al., 1997. JBC 272:8628-8634; Wu and Yung, 1994. Eur. J. Pharmacol. 270:203-212 α-Amanitin Baumann et al., 1994. Protein Sci. 3:750-756; Rudd and Luse, 1996. {\cs52\i JBC 271:21549-21558; Seiser et al., 1995. J. Biol. Chem. 270:29400-29406 Other compounds Brefeldin A Donaldson et al., 1991. J. Cell Biol. 112:579-588; Donaldson et al., 1992. Nature 360:350-352; Ktistakis et al., 1992. Nature 356:344-346; Lippincott-Schwartz et al., 1990. Cell 60:821-836 Cyclosporin A Su et al., 1997. J. Cell Biol. 139:1533-1543; Sugimoto et al., 1997. J. Biol. Chem. 272:29415-29418; McKeon, 1991. Cell 66:823-826; Nicolli et al., 1996. JBC 271:2185-2192 Standard Measurements, Data, and continued Abbreviations A.1K.5

Current Protocols in Molecular Biology Supplement 59 Table A.1K.1 Biological Activity of Drugs Commonly Used In Molecular Biological Research, continued Drug References Desferrioxamine Ben-Shachar et al., 1995. J. Neurochem. 64:718-723; Bergeron et al., 1996. J. Med. Chem. 39:1575-1581; Dang et al., 1994. Res. Commun. Mol. Pathol. Pharmacol. 86: 43-57; Denicola et al., 1995. Free Radical Biol. Med. 19:11-19; Henderson and Kuhn, 1995. JBC 270:20509-20515 2-Deoxyglucose Donaldson et al., 1991. J. Cell Biol. 112:579-588; Hill et al., 1998. JBC 273: 3308-3313; Villalba et al., 1994. JBC 269:2468-2476 Dithiothreitol Braakman et al., 1992. EMBO J. 11:1717-1722; Lodish and Kong, 1993. JBC 268:20598-20605; Simons et al., 1995. J. Cell Biol. 130:41- 49; Verde et al., 1995. Eur. J. Cell Biol. 67:267-274 Filipin Liu et al., 1997. JBC 272:7211-7222; Schnitzer et al., 1994. J. Cell Biol. 127:1217-1232; Silberkang et al., 1983. JBC 258:8503-8511; Smart et al., 1996. J. Cell Biol. 134:1169-1177; Stahl and Mueller, 1995. J. Cell Biol. 129:335-344

Fumonisin B1 Merrill et al., 1993. JBC 268:27299-27306; Sandvig et al., 1996. Mol. Biol. Cell. 7:1391-1404; Spiegel and Merrill, 1996. FASEB J . 10:1388- 1397; Wang et al., 1991. JBC 266:14486-14490; Wang et al., 1996. PNAS 93:3461-3465 Geneticin Canaani and Berg, 1982. PNAS 79:5166-5170; Southern and Berg, 1982. J. Mol. Appli. Genet. 1:327-341; Morris et al., 1996. JBC 271:15468-15477. Leptomycin B Fornerod et al., 1997. Cell 90:1051-1060; Fukuda et al., 1997. Nature 390:308-311; Wada et al., 1998. EMBO J. 17:1635-1641; Wolff et al., 1997. Chem. Biol. 4:139-147 Lovastatin Carel et al., 1996. JBC 271:30625-30630; Hancock et al., 1989. Cell 57:1167-1177; Jakobisiak et al., 1991. PNAS 88:3628-3632. Mendola and Backer, 1990. Cell Growth Differ. 1:499-502; Vincent et al., 1991. Biochem. Biophys. Res. Commun. 180:1284-1289 Lysophosphatidic acid An et al., 1998. JBC 273:7906-7910; Jalink et al., 1990. JBC 265:12232-12239; Moolenaar, 1995. JBC 270:12949-12952; Zhang et al., 1997. Mol. Biol. Cell. 8:1415-1425 Mastoparan Huber et al., 1997. J. Cell Sci. 110: 2955-2968; Klinker et al., 1996. Biochem. Pharmacol. 51:217-223; Konrad et al., 1995. JBC 270: 12869-12876; Schwaninger et al., 1992. J. Cell Biol. 119:1077-1096; Smith et al., 1995. JBC 270:18323-18328 Ouabain Croyle et al., 1997. Eur. J. Biochem. 248:488-495; Peng et al., 1996. JBC 271: 10372-10378; Swann and Steketee, 1989. J. Neurochem. 52:1598-1604 PDMP Chen et al., 1995. JBC 270:13291-13297; Inokuchi et al., 1987. Cancer Lett. 38:23-30; Maceyka and Machamer, 1997. J. Cell Biol. 139:1411-1418; Rosenwald et al., 1992. Biochemistry 31:3581-3590; Sandvig et al., 1996. Mol. Biol. Cell. 7:1391-1404; Uemura et al., 1990. J. Biochem. 108:525-530 Pertussis toxin An et al., 1998. JBC 273:7906-7910; Hewlett et al., 1983. Infect. Immun. 40:1198-1203; Jakobs et al., 1984. Eur. J. Biochem. 140:177- 181; Kopf and Woolkalis, 1991. Methods Enzymol. 195:257-266; Luttrell et al., 1997. JBC 272:31648-31656 Phorbol esters Janecki et al., 1998. JBC 273:8790-8798; Macfarlane and O’Donnell, 1993. Leukemia 7:1846-1851; Niedel et al., 1983. PNAS 80:36-40; Nishizuka, 1986. Science 233:305-312; Oishi and Yamaguchi, 1994. J. Compendium of Drugs Commonly Cell. Biochem. 55: 168-172; Schmidt et al., 1998. JBC 273:7413- Used in 7422; Tepper et al., 1995. PNAS 92:8443-8447 Molecular Biology Research continued A.1K.6

Supplement 59 Current Protocols in Molecular Biology Table A.1K.1 Biological Activity of Drugs Commonly Used In Molecular Biological Research, continued Drug References Rapamycin Brown et al., 1994. Nature 369:756-758 Jefferies et al., 1997. EMBO J. 16:3693-3704 Kozlovsky et al., 1997. J. Biol. Chem. 272:33367-33372 Liu et al., 1991. Cell 66:807-815 Sabers et al., 1995. J. Biol. Chem. 270:815-822 Sodium azide Bhat et al., 1996. JBC 271:32551-32556; Donaldson et al., 1991. J. Cell Biol. 112:579-588; van Klompenburg et al., 1997. EMBO J. 16:4261-4266 Sodium butyrate Calabresse et al., 1993. Biochem. Biophys. Res. Commun. 195:31-38; Gonzalez-Garay and Cabral, 1996. J. Cell Biol. 135:1525-1534; Russo et al., 1997. Biochem. Biophys. Res. Commun. 233:673-677; Vaziri et al., 1996. JBC 271:25921-25927; White et al., 1995. J. Cell Sci. 108:441-455 Trifluoperazine Aussel et al., 1995. JBC 270:8032-8036; Massom et al., 1990. Biochemistry 29:671-681; Rao, 1987. Biochem. Biophys. Res. Commun. 148:768-775 Valinomycin Inai et al., 1997. Cell Struct. Funct. 22:555-563; Loiseau et al., 1997. Biochim. Biophys. Acta. 1330:39-49; Orlov et al., 1994. FEBS Lett. 345:104-106; Szabo et al., 1997. JBC 272:23165-23171 W-7 de Figueiredo and Brown, 1995. Mol. Biol. Cell. 6:871-887; Hidaka et al., 1981. PNAS 78:4354-4357; Hunziker, 1994. JBC 269:29003- 29009; Wolf and Gross, 1996. JBC 271:20989-20992

DRUGS COMMONLY USED IN MOLECULAR BIOLOGY A23187 Calcium ionophore; forms stable complexes with divalent cations and increases their passage across biological membranes. Useful tool for increasing intracellular cal- cium concentration. The effectiveness of A23187 is dependent on the presence of extracellular calcium. Can be used as a fluorescent probe for investigating protein hydrophobicity. 4-Bromo-A23187 is a nonfluorescent derivative. Soluble in: DMSO, methanol Stock concentration: 100 mM (store at 4°C protected from light) Working concentration: 0.1 to 20 µM Duration of incubation: 2 min to 24 hr Aggregates over time in aqueous systems. Actinomycin D Inhibits transcription by complexing with deoxyguanosine residues on DNA and blocking the movement of RNA polymerase. A potent inducer of apoptosis in many cell lines. However, actinomycin D has also been shown to suppress programmed cell death of PC12 cells induced by the topoisomerase II inhibitor etoposide. Soluble in: Methanol Stock concentration: 100 mM (store at 4°C) Working concentration: 1 to 5 µM Duration of incubation: 5 min to 24 hr Standard Measurements, Data, and Abbreviations A.1K.7

Current Protocols in Molecular Biology Supplement 59 α-Amanitin Acts as a potent and specific inhibitor of mRNA synthesis by binding preferentially to RNA polymerase II. At high concentrations also inhibits RNA polymerase III. Soluble in: Methanol, water Stock concentration: 2 to 10 mg/ml (store at 4°C protected from light) Working concentration: 1 to 10 µg/ml (pol II) to 200 µg/ml (pol III) Duration of incubation: 15 to 60 min

Ammonium chloride (NH4Cl) Permeant weak base. Used to neutralize acidic endomembrane compartments. Inhibits synthesis of sphingoid bases. Soluble in: Water (freely soluble) Stock concentration: 5 M (store at 4°C) Working concentration: 1 to 50 mM Duration of incubation: Effective within 15 sec Anisomycin Inhibits protein synthesis by blocking the peptidyl transferase step during transla- tion. Activates p54 (JNK2) and MAP kinases. May be a useful tool to study cytoplasmic signals that result in nuclear signaling and c-fos and c-jun induction. Also known to induce apoptosis in U937 cells. Soluble in: DMSO Stock concentration: 100 µg/ml (store at 4°C) Working concentration: 50 ng/ml to 1 µg/ml. Duration of incubation: 30 min to 16 hr, depending on properties studied Aphidicolin Cell synchronization reagent. Reversible inhibitor of DNA polymerase α and δ; blocks cell cycle at early S phase. Potentiates apoptosis induced by arabinosyl nucleosides in leukemia cell lines. Soluble in: DMSO, methanol Stock concentration: 2 mg/ml (store at 4°C) Working concentration: 0.5 to 100 µg/ml. Duration of incubation: 12 to 24 hr Ara-C (cytosine arabinoside) Inhibits DNA synthesis. S-phase-toxic reagent whose active metabolite (ara-CTP) is a substrate for DNA polymerases and is incorporated into DNA. Anticancer, antiviral agent that is especially effective against leukemias. Induces apoptosis in human myeloid leukemia cells and in rat sympathetic neurons. Soluble in: Water Stock concentration: 20 mg/ml (store at 4°C) Working concentration: 0.1 to 1 µg/ml Duration of incubation: >3 hr

Bafilomycin A1 A potent and specific inhibitor of vacuolar-type H+-ATPases. Valuable tool for distinguishing different types of ATPases. Blocks lysosomal trafficking in macro- phages. Soluble in: DMSO Stock concentration: 50 µM (store at −20°C protected from light) Compendium of Drugs Commonly Working concentration: 10 to 100 nM Used in Duration of incubation: 10 min to 2 hr Molecular Biology Research A.1K.8

Supplement 59 Current Protocols in Molecular Biology BAPTA Ca2+ chelator with a 105-fold greater affinity for Ca2+ than for Mg2+; can be used to control the level of both intracellular (using its membrane-permeant AM ester) and extracellular Ca2+. Soluble in: DMSO Stock concentration: 1 to 10 mM (store at −20°C in aliquots, protected from light; avoid repeated freeze-thawing) Working concentration: (BAPTA-AM): 1 to 20 µM Duration of incubation: 15 to 60 min at 20° to 37°C Before incubation with BAPTA, wash cells 2 to 3 times with serum-free medium (serum may contain esterase activity). The cell-loading medium should also be free of amino acids or buffers containing primary or secondary amines that may cleave the AM esters and prevent loading. Bisindolylmaleimide I (GF 109203X) A highly selective cell-permeant protein kinase C (PKC) inhibitor that is structurally similar to staurosporine, but has higher selectivity. May inhibit protein kinase A at high concentrations. Acts as a competitive inhibitor for the ATP-binding site of the PKC catalytic domain. Since ATP levels are generally very high in cells, the potency of bisindolylmaleimide I is reduced accordingly in whole-cell assays. Soluble in: DMSO Stock concentration: 2 mM (store at ≤4°C) Working concentration: 20 nM to 1 µM Duration of incubation: 15 min to 6 hr Water-soluble salts are available. Brefeldin A Inhibits GTP nucleotide exchange onto several members of the ARF (ADP ribosy- lation factor) family. Inhibits binding of the cytosolic coatomer (COPI) complex to Golgi membranes; induces the rapid redistribution of the Golgi apparatus into the ER; blocks transport out of the ER in a number of cell lines. Reversible. Soluble in: Methanol Stock concentration: 1 to 20 mM (store at −20°C) Working concentration: 1 to 5 µM Duration of incubation: 5 min to 24 hr; effects are rapid (30 sec) 8-Bromo–cyclic AMP Cell-permeant cyclic AMP analog. Activates protein kinase A. Increased resistance to degradation by cellular phosphodiesterases as compared to cyclic AMP. Soluble in: Water Stock concentration: 100 mM (store at −20°C) Working concentration: 10 to 500 µM Duration of incubation: Up to 24 hr Calpain inhibitor I (ALLN) Inhibitor of calpain I, calpain II, cathepsin B, and cathepsin L. A peptide aldehyde, which inhibits neutral cysteine proteases and the proteosome. Protects against neuronal damage caused by hypoxia and ischemia. Inhibits proteolysis of IkB by

the ubiquitin-proteosome complex. Inhibits cell-cycle progression at G1/S and metaphase/anaphase in CHO cells by inhibiting cyclin B degradation. Membrane- permeant due to low molecular weight and lack of charged residues. Soluble in: DMSO, methanol, dimethylformamide Stock concentration: 25 mM (store at 4°C) Standard Measurements, Working concentration: 25 to 100 µM Data, and Duration of incubation: 1 to 18 hr Abbreviations A.1K.9

Current Protocols in Molecular Biology Supplement 59 Calphostin C

Potent and highly selective inhibitor of protein kinase C (PKC; Ki = 50 nM). Competes with phorbol esters and diacylglycerol for binding to the PKC regulatory domain. Does not compete with Ca2+ or phospholipids. At higher concentrations µ µ inhibits myosin light chain kinase (Ki > 5 M), protein kinase A (Ki > 50 M), protein µ v-src µ kinase G (Ki > 25 M), and p60 (Ki > 50 M). Soluble in: DMSO Stock concentration: 1 mM (store 4°C protected from light) Working concentration: 10 nM to 3 µM Duration of incubation: 15 to 60 min Brief exposure to visible light in the presence of PKC is required for PKC inhibition by calphostin C. See Table 1 (Selectivity of Selected Kinase Inhibitors) in the Biomol Catalog and Handbook, 5th ed., and Technical Note #11 from Alexis Biochemicals for additional information. Calyculin A Potent cell-permeant inhibitor with high specificity for the Ser/Thr protein phos- phatases 1 and 2A. Calyculin A is 20 to 300 times more potent than okadaic acid as a PP-1 class phosphatase inhibitor. Stimulates contraction of smooth muscle, induces intracellular protein phosphorylation in cultured human keratinocytes, and inhibits apoptosis. Soluble in: DMSO, ethanol Stock concentration: 10 µM (store at −20°C protected from light and moisture) Working concentration: 0.5 to 50 nM Duration of incubation: 15 min to 2 hr See Technical Note #19 from Alexis Biochemicals for additional information.May cause cell rounding. Camptothecin A reversible DNA topoisomerase I inhibitor. Induces breaks at replication forks by binding to and stabilizing the topoisomerase-DNA covalent complex. Causes S- phase cytotoxicity. Posesses anti-leukemic and anti-tumor properties. Inhibits tat- mediated transactivation of HIV-1. Soluble in: DMSO Stock concentration: 1 to 10 mM (store at 4°C) Working concentration: 0.1 to 10 µM Duration of incubation: Literature shows use from 15 min to 24 hr Castanospermine Inhibitor of α- and β-glucosidases; inhibitor of glycoprotein processing. Prevents calnexin and calreticulin binding to N-linked glycans on newly synthesized glyco- proteins. Inhibits HIV infectivity. Soluble in: Water Stock concentration: 1 mM (store at 4°C) Working concentration: 1 to 5 µM Duration of incubation: 15 min to 3 hr

Compendium of Drugs Commonly Used in Molecular Biology Research A.1K.10

Supplement 59 Current Protocols in Molecular Biology CCCP (carbonyl cyanide-m-chlorophenyl hydrazone) Proton ionophore. Uncoupling agent for oxidative phosphorlyation that inhibits mitochondrial function. Approximately 100 times as effective as 2,4-dinitrophenol at collapsing membrane potential. Inhibits transport processes and depresses growth. Soluble in: DMSO, ethanol Stock concentration: 1 to 10 mM (store at 4°C) Working concentration: 1 to 5 µM Duration of incubation: 5 to 15 min Chelerythrine chloride µ Potent, selective, cell-permeant inhibitor of protein kinase C (Ki = 0.66 M). Acts on the catalytic domain. Chelerythrine shows competitive kinetics with PKC sub- strates, but is not competitive with ATP. Thus, the high concentration of ATP within cells should not lower the potency of chelerytherine in whole cells as compared with that seen in purified enzyme preparations. Inhibits thromboxane formation and phosphoinositide metabolism in platelets. Induces apoptosis in HL-60 cells. Soluble in: DMSO Stock concentration: 10 mM (store at −20°C) Working concentration: 1 µM Duration of incubation: 15 min to 2 hr See Technical Note #8 from Alexis Biochemicals for additional information. Chloroquine Tertiary amine that accumulates within and neutralizes the pH of acidic organelles; various effects on phagosome-endosome and phagosome-lysosome fusion. Anti- malarial drug that works via carrier-mediated uptake in P. falciparum. May activate protein kinases. Soluble in: Water Stock concentration: 1 to 10 mg/ml (store at room temperature) Working concentration: 10 to 200 µg/ml Duration of incubation: 15 min to 2 hr Cholera toxin Contains a single A subunit (mol. wt. = 29 kDa) and a B subunit (mol. wt. = 55 kDa)

containing five B polypeptide chains. The B subunit binds to GM1 ganglioside receptors on the surface of cells and facilitates transport of the A subunit through the membrane. The A subunit catalyzes the ADP-ribosylation of an arginine residue α on the subunit of heterotrimeric G proteins (primarily Gs), reducing its intrinsic GTPase activity. Toxicity results from activation of membrane-bound adenylate cyclase. Consequently, increased intracellular cAMP levels result in increased electrolyte transport out of the cell and water loss. Cholera toxin requires ADP-ri- bosylation factor (ARF) for maximal activity. Soluble in: Water Stock concentration: 1 mg/ml (store at 4°C, do not freeze) Working concentration: 100 ng/ml to 2 µg/ml Duration of incubation: 2 to 24 hr

Standard Measurements, Data, and Abbreviations A.1K.11

Current Protocols in Molecular Biology Supplement 59 Colcemid Cell synchronization agent. Depolymerizes microtubules and limits microtubule formation. Low concentrations inactivate spindle dynamics. Induces apoptosis by blocking mitosis in HeLa S3 cells. Colcemid is a less toxic derivative of colchicine. Soluble in: Ethanol Stock concentration: 1 mM (store at or below room temperature) Working concentration: 1 to 10 µM Duration of incubation: 1 to 24 hr, depending on process studied Colchicine Inhibitor of mitosis, used in cell-division studies. Disrupts microtubules and inhibits tubulin polymerization. Induces apoptosis in PC 12 cells and in cerebellar granule cells. Soluble in: Ethanol Stock concentration: 1 mM (store at or below room temperature protected from light and moisture) Working concentration: 1 to 10 µM Duration of incubation: 1 to 24 hr, depending on process studied Concanamycin B + Highly specific and sensitive inhibitor of vacuolar-type H -ATPases (Ki = 20 pM). Related to concanamycin A (folimycin). More potent and specific than bafilomycin

A1. Inhibits acidification of organelles such as lysosomes and the Golgi apparatus. Blocks cell-surface expression of viral glycoproteins without affecting their synthesis. Soluble in: Methanol, ethanol Stock concentration: 10 µM (store at −20°C protected from light) Working concentration: 50 nM Duration of incubation: 5 min to 1 hr Cycloheximide Inhibits protein synthesis in eukaryotes but not prokaryotes. Blocks the translocation step during translation. Induces apoptosis in a number of cell types. However, it inhibits DNA cleavage in rat thymocytes treated with thapsigargin and ionomycin. Soluble in: Water, ethanol, methanol Stock concentration: 10 mg/ml (store at or below room temperature) Working concentration: 1 to 100 µg/ml, depending on cell type Duration of incubation: Effective within 10 min To achieve >90% inhibition of protein synthesis, only 1 to 10 ìg/ml is required in CHO and HeLa cells, but 100 ìg/ml is required in COS cells. Cyclosporin A (CsA) Cyclic oligopeptide with immunosuppressant properties. Induces apoptosis in some cell types, while inhibiting apoptosis in others. A complex of cyclosporin A and cyclophilin inhibits protein phosphatase 2B (calcineurin) with affinity at the nano- molar level. Inhibits nitric oxide synthesis induced by interleukin-1α, lipopolysac- charides, and TNFα. Soluble in: Ethanol, methanol Stock concentration: 1 to 5 mM (store at 4°C) Working concentration: 0.1 to 10 µM Duration of incubation: Used anywhere from 15 min to 24 hr Cytochalasin B Compendium of Drugs Commonly Cell-permeant fungal toxin that blocks the formation of contractile microfilaments. Used in Shortens actin filaments by blocking monomer addition at the barbed (fast-growing) Molecular Biology Research continued A.1K.12

Supplement 59 Current Protocols in Molecular Biology end of polymers. Inhibits cytoplasmic division, cell movement, phagocytosis, platelet aggregation, and glucose transport. Soluble in: DMSO, ethanol Stock concentration: 10 mM (store at −20°C protected from light) Working concentration: 1 to 20 µM Duration of incubation: 15 min to 2 hr Cytochalasin D Approximately 10-fold more potent than cytochalasin B in inhibiting actin filament function. Does not inhibit sugar transport in cells. Modulates CD4 cross-linking in T lymphocytes and increases intracellular Ca2+. Exhibits antitumor activity. Soluble in: DMSO Stock concentration: 10 mM (store at −20°C protected from light) Working concentration: 1 to 20 µM Duration of incubation: 15 min to 2 hr. Desferrioxamine (DFO) Iron-chelating agent. Commonly used in therapy as a chelator of ferric iron in iron overload disorders. Protects against dopamine-induced cell death. Also interferes with hydroxy-radical formation. Shows an antiproliferative effect on vascular smooth muscle cells. Soluble in: DMSO; slightly soluble in water Stock concentration: 10 to 50 mM (store at 4°C) Working concentration: 10 µM to 2 mM Duration of incubation: Up to 18 hr 2-Deoxyglucose Nonmetabolizable derivative of glucose. Competes with glucose for the GLUT-2 transporter; phosphorylation of 2-deoxyglucose by hexokinase effectively inhibits glucose flux through the glycolytic pathway. Used in combination with sodium azide or oligomycin to reduce cellular ATP levels. Blocks inhibition of IL-1 release by high glucose levels in RAW 264.7 cells. Soluble in: Water Stock concentration: 1 M (store at 4°C) Working concentration: 5 to 50 mM Duration of incubation: 15 min to 3 hr Deoxymannojirimycin Competitive α-mannosidase I inhibitor that blocks conversion of high mannose forms to complex oligosaccharides. Inhibits mammalian Golgi α-mannosidase I (an α-1,2-mannosidase). Other rat liver mannosidases are not significantly affected (α -1,2–specific ER mannosidase is only inhibited 2% to 5% by 100 mM deoxyman- nojirimycin, and Golgi α-mannosidase II is inhibited ∼14%). Soluble in: Water Stock concentration: 100 mM (store at −20°C) Working concentration: 1 to 5 mM Duration of incubation: Anywhere from 30 min to 24 hr Deoxynojirimycin Specific glucosidase inhibitor. Inhibits endoplasmic reticulum trimming glu- cosidases I and II, which sequentially remove three glucose residues from Glc Man GlcNAc in N-linked glycan biosynthesis. Prevents calnexin and cal- 3 9 2 Standard reticulin binding to N-linked glycoproteins within the ER. Measurements, Data, and continued Abbreviations A.1K.13

Current Protocols in Molecular Biology Supplement 59 Soluble in: Water Stock concentration: 100 mM (store at 4°C) Working conditions: 1 to 5 mM. Duration of incubation: 15 min to 24 hr At concentrations >1 mM, deoxynojirimycin may inhibit lipid-linked oligosaccharide bio- synthesis as well as trimming. In such cases, N-methyldeoxynojirimycin may be a more effective inhibitor, possibly owing to an increased ability to cross cell membranes, afforded by the N-methyl group. Dibutyryl cyclic AMP Highly membrane-permeant cAMP analog resistant to phosphodiesterase cleavage. Constitutive activator of protein kinase A. This product releases butyrate due to intracellular and extracellular esterase action. Butyrate may have its own distinct biological effects (see sodium butyrate). Soluble in: DMSO, ethanol Stock concentration: 1 M (store at −20°C) Working concentration: 100 µM to 1 mM Duration of incubation: Anywhere from 1 to 48 hr Dithiothreitol (DTT; Cleland’s reagent) Cell-permeant protective agent for SH groups; maintains monothiols completely in the reduced state and reduces disulfides quantitatively. DTT interferes with the folding and export of proteins located in the endoplasmic reticulum, but it does not prevent the transfer from the intermediate compartment to the Golgi complex. Reversible. Soluble in: Water, ethanol Stock concentration: 1 M (store at 4°C) Working concentration: 1 to 10 mM Duration of incubation: 1 min to several hours E-64 Irreversible inhibitor of cysteine proteases (papain and cathepsins B and L). Has no action on cysteine residues in other proteins. Soluble in: Water Stock concentration: 1 mg/ml (store at −20°C) Working concentration: 0.5 to 10 µg/ml Duration of incubation: Up to 24 hr Emetine Irreversibly blocks protein synthesis by inhibiting movement of ribosomes along mRNA. Stimulates rapid and differential phosphorylation of the stress-activated protein kinase/c-Jun kinase (SAPK/JNK) pathway. Prevents apoptosis in several cell lines. In primary rat hepatocytes, the relative potency of inhibition of several protein synthesis inhibitors is in the order: emetine > anisomycin > cycloheximide > puromycin, with puromycin exhibiting only marginal inhibition at a concentration of 1 µM. In fact, 90% to 95% inhibition of protein synthesis was achieved only with emetine and anisomycin, at 10 µM concentrations. Cycloheximide and puromycin exerted only 80% and 60% inhibition, respectively, at a similar concentration. Soluble in: Ethanol Stock concentration: 10 to 100 mM (store at 4°C protected from light) Working concentration: 10 to 20 µM Compendium of Drugs Commonly Duration of incubation: 15 to 30 min; can incubate 24 hr Used in Molecular Biology Research A.1K.14

Supplement 59 Current Protocols in Molecular Biology Etoposide (VP-16) Topoisomerase II inhibitor. Stabilizes the covalent complexes of topoisomerase II with DNA. Has major activity against a number of tumors, including germ cell neoplasms, small cell lung cancer, and malignant lymphoma. Induces apoptosis in mouse thymocytes and HL-60 cells. Activates PKCα. Soluble in: DMSO Stock concentration: 100 to 500 mM (store at room temperature) Working concentration: 50 to 200 µM Duration of incubation: 1 to 24 hr Filipin A cholesterol-binding fluorochrome. Specific for unesterified cholesterol. Binds and removes cholesterol from cell surface membranes. Reversibly disassembles caveolae. Soluble in: Methanol Stock concentration: 500 µg/ml (store 4°C protected from light) Working concentration: 5 to 50 µg/ml Duration of incubation: 1 hr Forskolin Activates adenylate cyclase by interacting directly with the catalytic subunit. Leads to an increase in the intracellular concentration of cAMP. Several forskolin deriva- tives are available having different and improved properties. Enhances detoxifica- tion of brefeldin A. Soluble in: DMSO, ethanol Stock concentration: 10 to 100 mM (store at 4°C) Working concentration: 10 µM (to increase cAMP levels); 100 µM (to inhibit bre- feldin A) Duration of incubation: Depending on assay, incubate cells 15 min to 12 hr

Fumonisin B1 Inhibits sphingolipid biosynthesis via inhibition of sphingosine N-acyltransferase (ceramide synthase). Sphingomyelin biosynthesis is preferentially inhibited versus glycosphingolipids in neuronal cells. Inhibits the butyric acid–induced increase in transport of cell-associated Shiga toxin to the Golgi apparatus and the ER. Induces apoptosis in monkey kidney cells. Soluble in: methanol Stock concentration: 10 to 100 mM (store at 4°C) Working concentration: Anywhere from 1 to 100 µM Duration of incubation: 15 min to 18 hr, depending on process studied Geneticin (G418) Aminoglycoside toxic to bacteria, yeast, higher plants, protozoa, and mammalian cells. Used for the selection and maintenance of eukaryotic cells stably transfected with the neomycin (neo) resistance genes from transposons Tn5 and Tn601. Soluble in: Water or culture medium Stock concentration: 2 mg/ml (active G418) in cell culture medium, adjust pH to ∼7.4 (store at 4°C) Working concentration: Usually 50 to 1000 µg/ml (optimal concentration must be determined experimentally and varies with the cell type used) Duration of incubation: >1 week Standard In cell types with relatively stable genomes (e.g., CHO), continuous incubation in geneticin Measurements, is not generally necessary once stable cells have been selected. Expression in stable cells Data, and with down-regulated viral promoters can be enhanced with sodium butyrate. Abbreviations A.1K.15

Current Protocols in Molecular Biology Supplement 59 Genistein Inhibits protein tyrosine kinases by acting as a competitive inhibitor of ATP. Prevents EGF-stimulated tyrosine phosphorylation in A431 cells, as well as inhibiting kinases in other cultured cells. Soluble in: DMSO Stock concentration: 100 to 500 mM (store at −20°C) Working concentration: 50 to 300 µM Duration of incubation: 15 min to 1 hr See Table II (Selectivity of Tyrosine Protein Kinase Inhibitors) in the Biomol Catalog and Handbook, 5th ed. H-7 A broad-based, cell-permeant serine/threonine kinase inhibitor. Inhibits protein kinase µ µ µ C (Ki = 6.0 M), protein kinase A (Ki = 3.0 M), protein kinase G (Ki = 5.8 M), and µ myosin light chain kinase (Ki = 97 M). Induces apoptotic DNA fragmentation and cell death in HL-60 cells. Numerous analogs with different selectivities are available. Soluble in: Water Stock concentration: 100 mM (store at 4°C) Working concentration: 10 to 100 µM Duration of incubation: 15 min to 3 hr See Table I (Selectivity of Selected Kinase Inhibitors) in the Biomol Catalog and Handbook, 5th ed., and Technical Note #10 from Alexis Biochemicals. Herbimycin A An irreversible and selective cell-permeant protein tyrosine kinase inhibitor; reacts with thiol groups. It is effective on Src, Yes, Fps, Ros, Abl, and ErbB oncogene products. Inhibits PDGF-induced phospholipase D activation in a dose-dependent manner. Soluble in: DMSO Stock concentration: 10 mM (store at −20°C protected from light) Working concentration: 1 to 10 µM Duration of incubation: 15 min to 1 hr Hydroxyurea Antineoplastic reagent. Blocks DNA synthesis by inhibiting ribonucleotide reduc- tase; accumulates cells at G1/S interface. Soluble in: Water Stock concentration: 1 M (store at 4°C) Working concentration: 50 µM to 1 mM Duration of incubation: Up to 24 hr Hygromycin Inhibitor of both prokaryotic and eukaryotic protein synthesis; inhibits at the translocation step on 70S ribosomes and causes misreading of mRNA. The E. coli hph hygromycin B–resistant gene is widely used for selection of recombinant clones in a variety of cell types. Hygromycin B is sold as an aqueous solution. Actual activity and concentration are given for each lot of product. Concentrations used must be experimentally determined. Ionomycin Ca2+ ionophore. Useful for increasing intracellular Ca2+ concentrations and in 2+ Compendium of measurement of cytoplasmic free Ca . More effective than A23187 and is nonfluo- Drugs Commonly rescent. Used in Soluble in: DMSO and methanol Molecular Biology Research Stock concentration: 1 mM (store at 4°C protected from light) continued A.1K.16

Supplement 59 Current Protocols in Molecular Biology Working concentration: Usually 1 µM Duration of incubation: 15 to 30 min. KN-62 2+ µ Potent and selective inhibitor of Ca /calmodulin kinase II (Ki = 0.9 M), displaying

a Ki for CaM kinase II more than 2 orders of magnitude lower than those for protein kinase C, protein kinase A, and myosin light chain kinase. A second inhibitor of CaM kinase II, KN-93, is more soluble in water and equally selective for CaM kinase µ II (Ki = 0.3 M). Prevents agonist-mediated activation of Ins(1,4,5)P3 3-kinase. Inhibits differentiation of 3T3-L1 embryonic fibroblasts to adipocytes. Inactive analogs are available. Soluble in: DMSO Stock concentration: 10 mM (store at 4°C) Working concentration: 2 to 10 µM Duration of incubation: 30 min (can incubate cells up to 48 hr) Lactacystin A cell-permeant and irreversible proteosome inhibitor. Blocks proteosome activity by targeting the catalytic β subunit. Induces neurite outgrowth in Neuro 2A mouse neuroblastoma cells and inhibits progression of synchronized Neuro 2A cells and

MG-63 human osteosarcoma cells beyond the G1 phase of the cell cycle. Inhibits NFκB activation. Has revealed the role of the proteosome in the degradation of many ER proteins. Soluble in: DMSO Stock concentration: 10 mM (store at −20°C) Working concentration: 10 to 20 µM Duration of incubation: 1 to 12 hr Latrunculin A Inhibits actin polymerization and disrupts microfilament organization as well as microfilament-mediated processes; 10 to 100-fold more potent than cytochalasins. Whereas cytochalasins induce dissolution of F-actin and stress-fiber contraction in fibroblasts in culture, the latrunculins (A and the less potent B) cause a shortening and thickening of stress fibers. In addition, the latrunculins sequester actin mono- mers, whereas with the cytochalasins, actin remains in an oligomer form. Thus, the two classes of compounds may have different target sites. Reversible. Soluble in: DMSO, ethanol Stock concentration: 10 mg/ml (store at −20°C) Working concentration: 0.2 to 10 µg/ml Duration of incubation: 1 to 12 hr Leptomycin B A potent and specific inhibitor of the NES-dependent nuclear export of proteins; binds to the export receptor CRM1. Exhibits antifungal and antitumor effects, inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 regulatory protein Rev, and exhibits significant antiproliferative activity. Soluble in: Ethanol Stock concentration: 100 µM to 1 mM (store at −20°C) Working concentration: 10 to 100 nM Duration of incubation: 30 min to 3 hr

Standard Measurements, Data, and Abbreviations A.1K.17

Current Protocols in Molecular Biology Supplement 59 Leupeptin A reversible inhibitor of trypsin-like and cysteine proteases (including trypsin, plasmin, proteinase K, papain, thrombin, and cathepsin A and B). Inhibits activa- tion-induced programmed cell death in T lymphocytes. Soluble in: Water Stock concentration: 1 to 10 mM (store at −20°C) Working concentration: 10 to 100 µM Duration of incubation: 15 min to several hours Lovastatin An antihypercholesterolemic agent and inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase; depletes endogenous pools of mevalonic acid, thereby blocking protein isoprenylation and cholesterol synthesis. Has a number of cellular effects. Blocks N-ras oncogene–induced neuronal differentiation, inhibits

growth factor signaling and causes cells to arrest in late G1 phase. Soluble in: DMSO, ethanol Stock concentration: 4 mg/ml (store at −20°C) Working concentration: 20 µM Duration of incubation: 6 to 24 hr Lysophosphatidic acid (LPA) Activates a number of signaling pathways and processes via heterotrimeric G

proteins (primarily Gi and Gq) including: inhibition of adenylate cyclase, activation of Ras and the Raf/MAP kinase pathway, stimulation of phospholipases C and D, and stress-fiber formation through the activation of Rho. Solubility: A stock solution may be prepared at 10 mg/ml in 95:5:5 chloro- form/methanol/acetic acid (gives a clear solution). Solubility in dimethysulfoxide (DMSO) or ethanol is limited. The sodium salt of oleoyl-LPA is reported to be readily soluble at 5 mg/ml (∼11 mM) in calcium and magnesium-free buffers (Jalink et al., 1990). Solubilization has also been achieved (Seufferlein and Rozengurt, 1994) in phosphate-buffered saline (PBS), pH 7.4, or calcium- and magnesium-free Dulbecco’s PBS (CMF-DPBS), pH 7.4 (see APPENDIX 2 for recipes), at up to 3 mM (0.14 mg/ml) in the presence of 0.1% (w/v) BSA (essentially fatty-acid free). Storage. LPA should be stable in solution under neutral conditions. Freezer storage is recommended for solutions or aqueous preparations. Maintaining the product under an inert atmosphere (nitrogen or argon) may be appropriate for some applications. Working concentration. Anywhere from 500 nM (1-hr incubation) to 100 µM (15-min incubation). LY294002 (PI 3-kinase inhibitor) Reversible inhibitor of phosphatidylinositol-3-kinase that acts on the ATP-binding site of the enzyme. Does not affect the activity of EGF receptor kinase, MAP kinase, PKC, PI4-kinase, S6 kinase, and c-src. Blocks proliferation of cultured rabbit aortic smooth muscle cells without inducing apoptosis. Wortmannin is more selective and more potent, but is irreversible. Soluble in: DMSO and ethanol Stock concentration: 1 to 10 mM (store in aliquots at −20°C) Working concentration: 1 to 2 µM Duration of incubation: 15 min to 3 hr Compendium of Drugs Commonly Used in Molecular Biology Research A.1K.18

Supplement 59 Current Protocols in Molecular Biology Mastoparan Relatively cell-permeant synthetic peptide capable of directly activating pertussis toxin–sensitive G proteins by a mechanism analogous to that of G-protein-coupled

receptors. Acts preferentially on Gi and Go rather than Gs. Stimulates insulin secretion in permeabilized cells, and can increase intracellular Ca2+ levels. Inhibits

calmodulin and activates phospholipase A2. Soluble in: Water Stock concentration: 1 mM (store at −20°C) Working concentration: 10 to 50 µM Duration of incubation: 15 min to 1 hr Microcystin-LR Cyclic heptapeptide; potent inhibitor of protein phosphatases 1 and 2A (PP-1 and

PP-2A). Unlike okadaic acid, microcystin-LR is equally effective on both PP-1 (Ki = 1.7 nM) and PP-2A (Ki = 0.04 nM). Has no effect on protein kinases, making it useful for reducing the effect of contaminating phosphatases in protein kinase assays. It is not cell-permeant, but can enter hepatocytes via the multispecific organic anion transporter. Soluble in: DMSO, ethanol, methanol Stock concentration: 1 mM (store at −20°C) Working concentration: 1 to 5 µM in hepatic cells; 10 µM in vitro Duration of incubation: 15 min to 1 hr MG-132 A potent, reversible and cell-permeant proteosome inhibitor. Reduces the degrada- tion of ubiquitin-conjugated proteins by the 26S complex without affecting its ATPase or isopeptidase activities. Has been used to implicate the proteosome in the breakdown of membrane proteins, including the CFTR, within the ER (see also lactacystin). Inhibits NFκB activation. Soluble in: DMSO Stock concentration: 10 to 100 mM (store at −20°C) Working concentration: 20 to 200 µM Duration of incubation: 30 min to 24 hr ML-7 (MLCK inhibitor)

Potent, cell- permeant, and selective inhibitor of myosin light chain kinase (Ki = 300 µ µ nM). Inhibits protein kinase A (Ki = 21 M) and protein kinase C (Ki = 42 M) at much higher concentrations. Soluble in: DMSO, ethanol, water Stock concentration: 100 to 500 mM (store at 4°C protected from light) Working concentration: 10 to 50 µM Duration of incubation: 15 min to 1 hr See Table I (Selectivity of Selected Kinase Inhibitors) in the Biomol Catalog and Handbook, 5th ed. L-Mimosine An inhibitor of DNA replication that may act by preventing the formation of replication forks. L-mimosine blocks the camptothecin-induced apoptosis of PC12 cells, whereas aphidicolin does not. Soluble in: Water Stock concentration: 10 mM (store at room temperature) Working concentration: 25 to 400 µM Standard Measurements, Duration of incubation: 2 to 24 hr Data, and Abbreviations A.1K.19

Current Protocols in Molecular Biology Supplement 59 Monensin Polyether antibiotic that functions as an Na+ ionophore. Forms stable complexes with monovalent cations that are able to cross cell membranes. Inhibits glycoprotein secretion by blocking transport through the Golgi. Neutralizes acidic endomem- brane compartments. Reduces sphingomyelinase activity. Soluble in: DMSO, methanol Stock concentration: 2 to 30 mM (store at 4°C) Working concentration: 1 to 30 µM Duration of incubation: Effective within seconds; use up to 3 hr. Nigericin Dual antiporter ionophore that acts as a K+/H+ exchanger. Stimulates Ca2+ release from mitochondrial stores by disruption of membrane potential. Allows adjustment of cytoplasmic pH (when combined with K+ ionophore such as valinomycin). Soluble in: Ethanol Stock concentration: 1 mg/ml (store at 4°C) Working concentration: 1 to 10 µM Duration of incubation: Effective within 2 to 5 min Nocodazole Has specific antimicrotubular activity for mammalian cells in culture. Promotes microtubule depolymerization. Nanomolar concentrations alter microtubule dy- namics and interfere with fibroblast locomotion without affecting polymer levels. Arrests cells in mitosis. Soluble in: DMSO Stock concentration: 10 to 30 mM (store at room temperature) Working concentration: 50 nM (low concentrations) to 30 µM (for effective mi- crotubule depolymerization) Duration of incubation: For rapid depolymerization of microtubules, preincubate cells on ice with nocodazole for 15 min; use up to 24 hr. Okadaic acid

Potent inhibitor of protein phosphatases, especially the PP-1 class (Ki = 10-15 nM) and PP-2A class (Ki = 0.1 nM), in numerous cell types. Does not affect the activity of acid or alkaline tyrosine phosphatases. It mimics the effects of insulin, enhances neurotransmitter release, causes vasodilation, and is a potent tumor promoter. Induces dispersal of the Golgi apparatus. Okadaic acid is a useful tool for studying cellular processes regulated by serine/threonine phosphorylation. Soluble in: DMSO, ethanol, methanol Stock concentration: 1 mM (store at −20°C protected from light) Working concentration: 50 to 200 nM Duration of incubation: 15 min to 2 hr May cause cell rounding. See Technical Note #18 from Alexis Biochemicals for additional information. Olomoucine Adenine derivative that acts as a competitive inhibitor for ATP binding and inhibits cdc2 µ p34 /cyclin B (Ki = 7 M) as well as several other CDKs at low concentrations. Does not significantly affect the activity of other protein kinases at 1 mM. Inhibits DNA synthesis in IL-2 stimulated T lymphocytes. Also used to synchronize cells in

Compendium of G1. Can affect microtubule dynamics at higher concentrations. Drugs Commonly Soluble in: DMSO Used in − ° Molecular Stock concentration: 100 mM (store in aliquots at 20 C) Biology Research µ µ Working concentration: Usually 10 M (up to 100 M) continued A.1K.20

Supplement 59 Current Protocols in Molecular Biology Duration of incubation: 15 min to 24 hr, depending on process studied. See Technical Note #25 from Alexis Biochemicals for additional information. Ouabain Selective Na+/K+-ATPase inhibitor. Causes net influx of Ca2+; also initiates the rapid protein kinase C–dependent inductions of early-response genes. Soluble in: Water Stock concentration: 100 mM (store at −20°C protected from light) Working concentration: 1 to 100 µM Duration of incubation: 30 min to 24 hr PDMP Useful tool for studying the effects of cellular glycosphingolipid depletion. Blocks ceramide glucosylation by inhibiting UDP-glucose:ceramide glucosyltransferase

(glucosylceramide synthetase). Has antitumor activity; arrests 3T3 cells at both G1/S and G2/M. Prevents sensitization of A431 cells to Shiga toxin. Slows the rate of both anterograde vesicular traffic and endocytosis in CHO and BHK-21 cells. Redistrib- utes cis-Golgi proteins to the ER. Soluble in: Ethanol Stock concentration: 10 to 100 mM (store at 4°C) Working concentration: 20 to 100 µM Duration of incubation: 1 to 18 hr, depending on process studied PD 98059 (MEK Inhibitor) Potent and selective inhibitor of MAP kinase kinase (MEK or MAPK/ERK kinase). Blocks the activity of MEK, thereby inhibiting the phosphorylation and activation of MAP kinase. Inhibits cell growth and reverses the phenotype of ras-transformed 3T3 mouse fibroblasts and rat kidney cells. Inhibits Golgi reassembly in vitro. Cell-permeant. Soluble in: DMSO Stock concentration: 50 mM (store at −20°C protected from light) Working concentration: 10 to 50 µM Duration of incubation: 30 min to 2 hr PMSF (phenylmethanesulfonyl fluoride) Inhibits serine proteases like chymotrypsin, trypsin, and thrombin, as well as acetylcholinesterase and the cysteine protease papain (reversible by DTT treatment). PMSF inhibits serine proteases by sulfonating serine residues at the active site. Does not inhibit metalloproteases, most cysteine proteases, or aspartic proteases. Soluble in: Anhydrous isopropanol at 35 mg/ml with heating, resulting in a clear to very slightly hazy, colorless to faint yellow solution, or in anhydrous (100%, not 95%) ethanol Stock concentration: 17 mg/ml (store at room temperature) Working concentration: 17 to 170 µg/ml Duration of incubation: 15 min to 1 hr PMSF is very unstable in the presence of water. The half-life of aqueous PMSF at 25°C at pH 7.0, 7.5, and 8.0 is 110, 55, and 35 min, respectively. Pepstatin A Inhibitor of aspartyl proteases, including pepsin, renin, cathepsin D, and HIV-1 protease. Inhibits degradation of ApoB in rat hepatocytes; inhibits cytokine-induced programmed cell death. Accelerates amyloid fibril formation in mice. Standard Soluble in: DMSO Measurements, − ° Data, and Stock concentration: 10 to 35 mM (store at 20 C) Abbreviations A.1K.21

Current Protocols in Molecular Biology Supplement 59 Working concentration: 50 to 100 µM in cells Duration of incubation: 30 min to 2 hr Pertussis toxin Protein endotoxin that catalyzes ADP-ribosylation of GDP-bound α subunits of the

G proteins Gi, Go, and Gt. Uncouples G proteins from receptors, thereby keeping the G protein in the inactive state. Used in the study of adenylate cyclase regulation

and the role of Gi proteins. Consists of an enzymatically active A protomer subunit (S-1) which posesses both NAD+ glycohydrolase and ADP-ribosylation activities and a B oligomer subunit (S-2, S-3, S-4, and S-5) that is responsible for cell surface attachment. Stock solution: Reconstitute commercial preparation in water. Generally, commer- cially available pertussis toxin (Alexis Biochemicals, Biomol, Calbiochem) contains 50 µg of protein in 10 mM sodium phosphate buffer, pH 7.0/50 mM sodium chloride after being resuspended in 0.5 ml water. It is an insoluble pro- tein that should be shaken gently before use. Store stock solutions at 4°C. Do not freeze. Working concentration: 50 to 100 ng/ml Duration of incubation: 2 to 24 hr Phenylarsine oxide (PAO) µ A cell-permeant phosphotyrosine phosphatase inhibitor (Ki = 18 M). Induces a dose-dependent increase in the free Ca2+ intracellular concentration in rat peritoneal macrophages, human foreskin fibroblasts, and cultured human endothelial cells, without affecting intracellular stores. Inhibits insulin activation of phosphatidyli- nositol 3′-kinase. Dithiol cross-linking agent. Soluble in: DMSO and chloroform Stock concentration: 50 mM (store at room temperature) Working concentration: 10 to 50 µM Duration of incubation: Anywhere from 15 sec to 2 hr Phorbol esters An example is phorbol myristate acetate (PMA). Extremely potent tumor promoters. Activate protein kinase C by mimicking diacylglycerols (DAGs), causing a wide range of effects in cells and tissues. Soluble in: DMSO Stock concentration: 1 mM (store at −20°C) Working concentration: 50 nM to 3 µM Duration of incubation: Cells can be incubated anywhere from 5 min to 48 hr; stable in cells; results in long-term activation of PKC. However, long-term treatment may cause down-regulation of certain PKC subtypes. See Technical Notes #13 and #14 from Alexis Biochemicals for additional information. Piceatannol At low concentrations, inhibits the receptor-mediated activation of the protein tyrosine kinase Syk as compared to the Src family in mast cells and B cells. Inhibits FcεR1-mediated signaling in RBL-2H3 cells. Soluble in: DMSO, ethanol Stock concentration: 10 to 50 mg/ml (store at 4°C protected from light) Working concentration: 10 to 30 µg/ml Compendium of Duration of incubation: 1 hr Drugs Commonly Used in Molecular Biology Research A.1K.22

Supplement 59 Current Protocols in Molecular Biology Puromycin Protein synthesis inhibitor. Causes premature release of nascent polypeptide chains by its addition to the growing chain end; structural analog of aminoacyl-tRNA. Soluble in: Water Stock concentration: 100 mM (store at −20°C) Working concentration: 10 to 100 µM Duration of incubation: 5 min to 1 hr Rapamycin Member of a family of macrolide immunosuppressants that binds to and inhibits the peptidylproline cis-trans isomerase (PPIase) activity of the immunophilin FKBP12; effectors include a large protein termed FRAP (FKBP12 rapamycin-associated protein). FKBP12-rapamycin binds to but does not inhibit the activity of the Ca2+/calmodulin–dependent serine/threonine phosphatase calcineurin. Blocks sig- naling, leading to the activation of p70 S6 kinase. Soluble in: DMSO, methanol, ethanol Stock concentration: 2 mM (store at −20°C) Working concentration: 1 to 20 nM Duration of incubation: 30 min to 1 hr

Sodium azide (NaN3) Inhibits mitochondrial ATPases; generally used to deplete ATP levels within cells (often in combination with 50 mM 2-deoxyglucose). Soluble in: Water Stock concentration: 1 M (store at room temperature) Working concentration: 10 to 20 mM Duration of incubation: 15 to 90 min Sodium butyrate A physiologically produced short-chain fatty acid that is generally used to increase expression of transfected genes with viral promoters (inhibits histone deacetyla-

tion). Blocks serum-stimulated DNA synthesis via a G1 block. Induces apoptosis in colon carcinoma cell lines by a p53-independent process. Interferes with signal- transduction processes, including the release of Ca2+ from intracellular stores. Soluble in: Water Stock concentration: 5 M (store at –20o C) Working concentration: 2 to 5 mM Duration of incubation: Usually >12 hr Sodium orthovanadate Broad-spectrum inhibitor of protein tyrosine phosphatases. Also inhibits other ATPases, by mimicking the γ phosphate of ATP, including Na+/K+ ATPase, acid and alkaline phosphatases, and adenylate cyclase. Vanadate is also a strong inhibitor of lysosomal proteolysis in hepatocytes, the effect being ascribed to a direct inhibition of lysosomal enzymes. Stimulates pp60 (v-src) kinase activity in intact cells. It also stimulates amino acid transport activity in skeletal muscle, in a rapid and concen- tration-dependent manner. 3− Simple aqueous solutions of (VO4) ion involve a dozen or more ionic species, both 3− monomeric and oligomeric, whose abundances depend upon pH and [VO4] con- centration. See Fohr et al. (1989) for directions on preparing monomeric or- thovanadate. It is unclear how readily vanadate ions enter cells (likely through anion transporters). At the concentration required for maximum inhibition, vanadate may Standard Measurements, have side effects that limit its application in cell culture. Can be combined with Data, and continued Abbreviations A.1K.23

Current Protocols in Molecular Biology Supplement 59 hydrogen peroxide (forming peroxyvanadate) to facilitate cell entry (combine 100 µ µ µ l 0.1 M orthovanadate, 900 l water, and 3.3 l 30% H2O2; use 1:100 dilution of this on cells). However, the effect of hydrogen peroxide itself should be tested. Soluble in: Water Stock concentration: 100 mM (store at room temperature). To ensure the presence of monomers, the solution is heated to boiling until translucent and the pH is readjusted to 10. Solutions can be divided into aliquots, stored in plastic, and frozen. The orange color observed before boiling is due to decavanadate. At pH 10 this will slowly depolymerize over several hours to the colorless monovanadate. Vanadyl, metavanadate, orthovanadate, and decavanadate will interconvert in aqueous solu- tion without suitable precautions (i.e., control of pH, oxidation state, complexing compounds, and concentration). Working concentration: 200 µM to 2 mM Duration of incubation: 15 min to 2 hr Staurosporine A potent cell-permeant inhibitor of protein kinases, most potently protein kinase C

(Ki = 0.7 nM), protein kinase A (Ki = 7 nM), and myosin light chain kinase (Ki = 1.3 nM). Interaction is with the ATP binding site. Induces apoptosis, but not DNA

fragmentation in MCF-7 cells. Arrests normal cells at the G1 checkpoint. Soluble in: DMSO, methanol Stock concentration: 1 mM (store at −20°C protected from light) Working concentration: 10 to 200 nM Duration of incubation: 30 min to 24 hr, depending on the assay used See Table I (Selectivity of Selected Kinase Inhibitors) in the Biomol Catalog and Handbook, 5th ed. Taxol (Paclitaxel) Antitumor and antileukemic agent. Promotes assembly of microtubules and inhibits microtubule disassembly. Bundles microtubules after several hours. Similar to nocodazole, taxol can inhibit microtubule dynamics without affecting overall poly-

mer levels at nanomolar concentrations. Blocks cells at the G2/M stage. Induces apoptosis in several cell types. Soluble in: DMSO, methanol Stock concentration: 20 mM (store at −20°C protected from light) Working concentration: 10 nM to 20 µM Duration of incubation: Taxol works rapidly to stabilize microtubules (within sev- eral minutes), although bundling takes several hours (this may be facilitated by first depolymerizing the polymer pool with ice treatment and/or washout of low levels of nocodazole) Thapsigargin Potent inhibitor of sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER) Ca2+- 2+ ATPases. Induces IP3-independent release of Ca from the endoplasmic reticulum, causing an increase in intracellular Ca2+. Depletion of Ca2+ from intracellular stores induces stress response and defects in protein folding and processing. Induces apoptosis in rat thymocytes and in human hepatoma cells. Irreversible. Soluble in: DMSO, ethanol Stock concentration: 1 mM (store at −20°C in aliquots, protect from light) µ Compendium of Working concentration: 20 nM to 1 M Drugs Commonly Duration of incubation: 15 sec to 2 min produces rise in intracellular Ca2+; Used in Molecular longer incubations may be used, depending on effect to be analyzed Biology Research See Technical Note #15 from Alexis Biochemicals for additional information. A.1K.24

Supplement 59 Current Protocols in Molecular Biology Trifluoperazine Calmodulin antagonist. At 10 µM, potentiates rise in cytosolic calcium induced by agonists. Antagonizes calmodulin at higher concentrations. Inhibits IL-2 production in activated Jurkat T cells. Structurally distinct from W-7. Soluble in: Water (dihydrochloride salt) Stock concentration: 10 mM (store at 4°C) Working concentration: 10 to 50 µM Duration of incubation: 10 min to 3 hr Tunicamycin Nucleoside antibiotic that inhibits N-linked glycosylation, specifically by blocking the transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamine to dolichol monophosphate; has no effect on other glycosylation forms, such as Ser/Thr-linked oligosaccharides. Causes misfolding and retention of numerous glycoproteins in the endoplasmic reticulum, which induces synthesis of ER chaperones. Soluble in: DMSO, ethanol Stock concentration: 10 mg/ml (store at −20°C) Working concentration: 1 to 10 µg/ml Duration of incubation: Cells can be treated from 1 to 24 hr Tyrphostins Large family of protein tyrosine kinase inhibitors. Inhibits receptors such as EGF receptor and PDGF receptor. Soluble in: DMSO, ethanol Stock concentration: 20 to 100 mM (store at −20°C protected from light) Working concentration: 10 to 150 µM Duration of incubation: Anywhere from 1 to 48 hr See Technical Note #22 from Alexis Biochemicals for additional information. Also see Table II (Selectivity of Tyrosine Protein Kinase Inhibitors) in the Biomol Catalog and Handbook, 5th ed. Valinomycin Potassium ionophore. Decreases ATP synthesis by decreasing membrane potential at mitochondrial membranes. Reported to inhibit NGF-induced neuronal differen- tiation. Used with nigericin to adjust cytoplasmic pH. Soluble in: DMSO Stock concentration: 1 mM (store at room temperature) Working concentration: 1 to 20 µM Duration of incubation: Normally 15 min to 2 hr Vinblastine Vinca alkaloid; antitumor drug. Inhibitor of cell proliferation that acts by disrupting spindle microtubule function. Binds tubulin and suppresses microtubule dynamics. Depolymerizes microtubules at higher concentrations. Induces apoptosis in cultured hepatocytes and human lymphoma cells. Similar, but not identical effects observed with another vinca alkaloid, vincristine. Soluble in: Methanol Stock concentration: 20 mM (store at 4°C protected from light) Working concentration: <10 nM (suppresses microtubule dynamics); 100 nM to 1 µM (depolymerizes microtubules); >10 µM (forms non-microtubule poly- mers) Standard Duration of incubation: 30 min to 24 hr Measurements, Data, and Abbreviations A.1K.25

Current Protocols in Molecular Biology Supplement 59 W-7 Member of a family of calmodulin antagonists, inhibiting calcium/calmodulin regulated enzyme activity. W-7 inhibits the Ca2+/calmodulin–induced activation of µ µ myosin light chain kinase (Ki = 51 M) and phosphodiesterase (Ki = 28 M). Inhibits membrane tubulation in cells treated with brefeldin A. Soluble in: Water Stock concentration: 10 to 100 mM (store at 4°C protected from light) Working concentration: 10 to 100 µM Duration of incubation: 30 min to 2 hr Wortmannin Selective and potent phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor; forms covalent associations with the kinases and is, therefore, irreversible. Abolishes

PDGF-mediated Ins(3,4,5)P3 formation in fibroblasts. Blocks the metabolic effects of insulin in isolated rat adipocytes without affecting the insulin receptor tyrosine kinase activity. Inhibits the formation of constitutive transport vesicles from the TGN. In human fetal undifferentiated cells, wortmannin induces morphological and functional endocrine differentiation. Soluble in: DMSO Stock concentration: 1 to 20 mM (store at −20°C in aliquots, protected from light) Working concentration: 10 to 100 nM Duration of incubation: 30 min to 4 hr At nanomolar concentrations, wortmannin is specific to PI 3-kinases, while at higher concentrations other kinases are affected. Once diluted into aqueous solutions, wortmannin is less stable and should be made fresh daily.

LITERATURE CITED Fohr, K.J., Scott, J., Ahnert-Hilger, G., and Gratzl, M. 1989. Characterization of the inositol 1,4,5-trisphos- phate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanadate and p-hydroxymercuribenzoate. Biochem. J. 262:83-89. Jalink, K., van Corven, E.J., and Moolenaar, W.H. 1990. Lysophosphatidic acid, but not phosphatidic acid, is a potent Ca2+-mobilizing stimulus for fibroblasts. Evidence for an extracellular site of action. J. Biol. Chem. 265:12232-12239. Seufferlein, T. and Rozengurt, E. 1994. Lysophosphatidic acid stimulates tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130: Signaling pathways and cross-talk with platelet-derived growth factor. J. Biol. Chem. 269: 9345-9351.

Contributed by Nelson B. Cole University of Pennsylvania Philadelphia, Pennsylvania

Compendium of Drugs Commonly Used in Molecular Biology Research A.1K.26

Supplement 59 Current Protocols in Molecular Biology